Neutrophils are the most prevalent immune cells in the synovial fluid in inflamed joints of children with oligoarticular juvenile idiopathic arthritis (JIA). Despite this, little is known about neutrophil function at the site of inflammation in JIA and how local neutrophils contribute to disease pathogenesis. This study aimed to characterize the phenotype and function of synovial fluid neutrophils in oligoarticular JIA.n = 17) and functionally  by flow cytometry. In a subset of patients (n = 6), blood samples were also obtained during inactive disease at a follow-up visit. The presence of CD206-expressing neutrophils was investigated in synovial biopsies from four patients by immunofluorescence.Neutrophils obtained from paired blood and synovial fluid from patients with active oligoarticular JIA were investigated phenotypically (hi CD62Llowaged phenotype. A large proportion of the synovial fluid neutrophils expressed CD206, a mannose receptor not commonly expressed by neutrophils but by monocytes, macrophages, and dendritic cells. CD206-expressing neutrophils were also found in synovial tissue biopsies. The synovial fluid neutrophil phenotype was not dependent on transmigration alone. Functionally, synovial fluid neutrophils had reduced phagocytic capacity and a trend towards impaired oxidative burst compared to blood neutrophils. In addition, the effector functions of the synovial fluid neutrophils correlated negatively with the proportion of CD206+ neutrophils.Neutrophils in synovial fluid had an activated phenotype, characterized by increased CD66b and CD11b levels, and most neutrophils had a CD16Neutrophils in the inflamed joint in oligoarticular JIA were altered, both regarding phenotype and function. Neutrophils in the synovial fluid were activated, had an aged phenotype, had gained monocyte-like features, and had impaired phagocytic capacity. The impairment in phagocytosis and oxidative burst was associated with the phenotype shift. We speculate that these neutrophil alterations might play a role in the sustained joint inflammation seen in JIA. Juvenile idiopathic arthritis (JIA) is an inflammatory rheumatic joint disease affecting children. Despite disease onset being at a young age, symptoms may be lifelong and include irreversible joint damage or growth disturbances , 2. The Previously, the immunopathogenesis of oligoarticular JIA has been thought to be driven primarily by adaptive immune responses, as the disease is associated with HLA class II genetic variants and the presence of autoantibodies . HoweverNeutrophils represent a major part of the innate immune system, and the pathology of several autoimmune rheumatic diseases are at least partly driven by dysfunctional neutrophils , 13. NeuThe major neutrophil effector functions include phagocytosis and oxidative burst. Neutrophil phagocytosis is important not only as a defense against infection but also for clearance of potential autoantigens. Reactive oxygen species (ROS) were long considered harmful byproducts in sterile inflammation but are now recognized for their immunosuppressive properties, and impaired ROS production is associated with rheumatic disease both in patients and animal models of disease , 17–23. The phenotype of synovial fluid neutrophils in JIA is insufficiently characterized, and two contradicting studies describe them as being highly activated  or restiIn this study, we set out to investigate both phenotype and effector functions of neutrophils in paired blood and synovial fluid from children with oligoarticular JIA. To characterize several important aspects of neutrophil biology, we investigated neutrophil phenotype by surface marker expression related to activation, tissue migration, maturity, and monocyte-like phenotype. Neutrophil function was analyzed in regard to both phagocytosis and ROS production. We hypothesized that synovial fluid neutrophils would differ from circulating neutrophils both regarding phenotype and function and that the neutrophil alterations can be important in driving and maintaining the local joint inflammation in oligoarticular JIA.n = 17, except for one patient (no. 5) who had received intraarticular steroids in another joint, 2 months prior to sampling. The blood and synovial fluid  during active arthritis were collected from all 17 patients. Synovial tissue biopsies were obtained from four patients, one of which is not included in phenotype or function analyses due to ongoing treatment with methotrexate (patient no. 18), in conjunction with synovial fluid aspiration. Follow-up blood samples from periods of inactive disease, defined as no signs of arthritis and/or uveitis at clinical examination, were collected from six of the patients. During periods of inactive disease, some of the patients were treated with conventional or biologic disease-modifying antirheumatic drugs (DMARD). Patient characteristics are described in Table All children fulfilling the International League of Associations for Rheumatology (ILAR) criteria for persistent oligoarticular JIA undergoing therapeutic arthrocentesis between 2016 and 2019 were offered to participate in this study. We included patients who were untreated or solely on non-steroid anti-inflammatory drugs (NSAIDs) for at least 6 months Healthy controls were included after informed consent. All controls were adults and contributed with blood and oral samples.Blood, synovial fluid, and oral samples were processed and analyzed immediately after collection.g, 10 min to pellet the cells. Synovial cells were counted and resuspended in phosphate-buffered saline (PBS) at a final concentration of 1 × 106/ml for use in flow cytometric staining. The synovial fluid supernatant was transferred to a new tube and centrifuged a second time at 800g, 10 min to pellet potential remaining cells or debris. The cell-free synovial fluid was collected and stored at − 80 °C.Synovial fluid was centrifuged at 400Equal volumes of blood, synovial cell suspension, or oral cell suspension were stained with antibody panels described in Supplemental Figure hi and CD62Lhi, immature neutrophils are CD16mid and CD62Lhi, and aged neutrophils are CD62Llow [Neutrophil maturity was determined using surface markers CD62L and CD16, where mature neutrophils are CD16CD62Llow –30. ActiParaffin-embedded biopsies in 3-μm sections mounted on microscope glass slides were baked for 1 h at 60 °C followed by deparaffinization in Neo-Clear (Merck) and a series of washes in ethanol of decreasing concentration. Antigen retrieval was performed in citrate buffer pH 6.0. The slides were blocked with 5% bovine serum albumin (BSA) and 5% normal goat serum (Abcam) for 30 min at room temperature prior to staining with primary antibodies rabbit anti-human MPO 1:200 (Dako A0398) and mouse anti-CD206 1:200 (Antibodies-Online clone:22–130) in PBS with 1% BSA at 4 °C overnight. After PBS washes, the slides were stained with secondary antibodies goat anti-rabbit Alexa Fluor 488 (Invitrogen) and donkey anti-mouse Alexa Fluor 568 (Abcam). The slides were mounted with Prolong Gold AntiFade with DAPI (Invitrogen). Images were taken using a Zeiss fluorescence microscope and processed in the Fiji software.2O. The purified neutrophils, 1 × 106/ml, were incubated in MCDB 131 medium (Gibco) with 20% synovial fluid or 20% serum from a blood donor at 37 °C for 1 h. Following incubation, neutrophils were washed with PBS and phenotypically analyzed by flow cytometry.Neutrophils were isolated from heparinized whole blood from healthy controls using Lymphoprep (Axis-Shield) density gradient centrifugation according to the manufacturer’s instructions followed by sedimentation of red blood cells in saline with 1.5% Dextran T500 (Pharmacosmos). Neutrophils in the supernatant were collected and pelleted, and contaminating red blood cells were lysed with sterile Hg, 5 min. The pellet was resuspended in PBS with 0.1% BSA and 2 mM EDTA. Epithelial cells were removed by incubation of biotinylated anti-E-cadherin (Bioss) at a final concentration of 4 μg/ml for 30 min followed by streptavidin-Dynabeads M280 (Invitrogen) and removal by a magnet. The remaining cells were washed with PBS/BSA/EDTA. Leukocytes were isolated using DSB-X™ biotin-labeled anti-CD45 at 2 μg/ml  and collected with Dynabeads FlowComp™ beads on a magnet. Cells were released from the beads using FlowComp™ release buffer, pelleted, and resuspended in PBS.Healthy volunteers performed a mouth rinse with 5 ml 0.9% NaCl for 30 s in the morning, prior to breakfast and toothbrushing. The collected rinse fluid was filtered through 10-μm syringe Filcons (BD Biosciences) and centrifuged at 300Escherichia coli and phagocytosis of fluorescently labeled opsonized E. coli respectively. When indicated, 90 μl serum or synovial fluid was added to 10 μl whole blood before analysis.Phagocytosis and oxidative burst were analyzed using Phagoburst™ and Phagotest™ assay kit (BD Biosciences) according to the manufacturer’s instructions. Both methods are flow cytometric assays performed on 100 μl heparinized whole blood or synovial fluid, measuring ROS production upon stimulation with phorbol-myristate-acetate (PMA) or opsonized U test. Correlations were analyzed using the Pearson r correlation coefficient. Statistical analyses were performed in Prism 9.Paired samples were analyzed using Wilcoxon’s matched-pairs signed-rank test. Comparisons between groups of independent data were analyzed using the Mann-Whitney hi, CD62Lhi), with very few immature  or aged (CD62Llow) neutrophils. However, in the synovial fluid, most neutrophils had an aged phenotype , and no immature neutrophils were observed . Neutrophils co-expressing both MPO and CD206 were observed in tissue biopsies from patients 4, 8, and 18  and increased CD10 compared to neutrophils in the blood. The high amount of neutrophils with an aged phenotype in the joint could be due to the anti-apoptotic effects of synovial fluid and inflammatory stimulation [low and hypersegmented phenotype [low neutrophils seen in the synovial fluid could be a consequence of both aging and/or activation and tissue migration. Neutrophils may become activated by a wide variety of stimuli, such as inflammatory cytokines and chemotactic stimulation [In a healthy joint, the synovial fluid normally contains no or few immune cells. During arthritis, immune cells are recruited from the bloodstream by chemotactic stimuli. Neutrophils found in the synovial fluid have extravasated from the synovial blood vessels and migrated through the synovial tissue that contains both synovial fibroblasts and infiltrating immune cells. In our study, the majority of these transmigrated neutrophils in the synovial fluid had an aged phenotype, characterized by low surface levels of mulation , 33. Thehenotype . Howeverhenotype . Thus, tmulation , and act+ neutrophils are also described in a few publications of mouse models of stroke and myocardial infarction, where they are called “N2” neutrophils in analogy with M2 macrophages [+ neutrophils in synovial biopsies where we found that some, but not all, of the neutrophils scattered within the synovial tissue had expression of CD206. The CD206 mannose receptor recognizes mannosylated and sulfated sugars and has a wide variety of functions including phagocytosis and promotion of antigen presentation [A large proportion of the synovial fluid neutrophils expressed the mannose receptor CD206, a receptor usually expressed by monocytes, macrophages, and dendritic cells, which is upregulated on synovial fluid monocytes in JIA . In macrrophages –39. In orophages . We confentation .+ neutrophils and increased levels of the TLR4 co-receptor CD14. Neutrophils expressing surface markers related to other myeloid phenotypes have previously been found in the synovial fluid in JIA and RA. In the recent study of JIA synovial fluid neutrophils by Metzemaekers et al., a significant portion of the synovial fluid neutrophils had gained expression of HLA-DR [It seems as if the synovial fluid neutrophils are gaining a phenotype with features of both the neutrophil and monocyte lineages, based on the large proportion of CD206f HLA-DR , and synf HLA-DR , 26. As f HLA-DR , it is pf HLA-DR –44, and The phenotype shift seen in synovial fluid neutrophils is not dependent on either synovial fluid or transmigration separately. In vitro exposure of JIA synovial fluid to healthy blood neutrophils did not cause a phenotype shift. We further studied oral cavity neutrophils as a control for tissue migration towards a non-inflammatory site. The synovial fluid and oral cavity are very different, where the oral cavity for instance carries a microflora while the synovial fluid is sterile, but still, we found that neutrophils at both sites shared some common features . However, the extent of the alterations was different in the two locations, and other changes occurred in only one of the sites . The findings of distinct phenotypes varying between the synovial fluid and oral cavity are supported by previous publications studying neutrophils in the synovial fluid compared to skin blister fluid or pleural effusions . Our resImportantly, we found a significant decrease in phagocytic capacity of the synovial fluid neutrophils compared to circulating neutrophils. An impaired neutrophil function may drive inflammation and autoimmunity, as neutrophil clearance of debris and dead cell remnants is essential to maintain homeostasis and minimize the autoantigenic burden . Extrace+ neutrophils might indicate that the neutrophils are becoming monocyte-like in regard to both phenotype and function. We speculate that the shift in neutrophil phenotype and function between the blood and synovial fluid in oligoarticular JIA could be an interesting target for the development of future therapies.Neutrophil activation or priming is often associated with increased effector functions , while olow neutrophils. Neutrophil function is not limited to oxidative burst and phagocytosis, and to further characterize the functional differences between circulating and synovial fluid neutrophils in JIA including chemotaxis, the release of neutrophil extracellular traps (NETs) and acquired ability to present antigens would be of interest and importance. These possible functional alterations will be investigated in further studies.The neutrophil gating strategy for patient neutrophils and the fact that more aspects of synovial fluid neutrophil effector functions were not investigated present some limitations to the study. The panels were designed for negative selection of neutrophils, but as synovial fluid may contain cells and particles with neutrophil size and granularity without leukocyte surface markers, a positive selection marker was also needed. After negative selection, neutrophils were therefore selected by CD16 positivity, which may inadvertently lose immature CD16This study demonstrates that neutrophils in the synovial fluid of children with active oligoarticular JIA are altered both phenotypically and functionally compared to paired neutrophils from the circulation. We observed that synovial fluid neutrophils are activated, aged, and has gained a monocyte-like phenotype, which is associated with an impairment in neutrophil phagocytosis and oxidative burst. We suggest that these neutrophil alterations could be of importance in sustaining joint inflammation and thus interesting possible targets for specific therapies.Additional file 1 : Supplemental table I. Absolute leukocyte counts and relative neutrophil frequencies in blood and synovial fluid. Additional file 2 : Supplemental Figure 1. Neutrophil gating strategies. Supplemental Figure 2. Neutrophil surface markers. Supplemental Figure 3. Patient age does not influence neutrophil phagocytosis or oxidative burst.


Ureaplasma spp. with a lower burden than those with intra-amniotic infection (p = 0.03). The intensity of intra-amniotic inflammatory response measured by interleukin-6 was higher in women with colonization than in those with negative amniotic fluid . Women with colonization had higher rates of acute inflammatory placental lesions than those with negative amniotic fluid. In PPROM, colonization, caused mainly by microorganisms from the lower genital tract, might represent an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response.To determine the main clinical characteristics of preterm prelabor rupture of membranes (PPROM) complicated by colonization of the amniotic cavity . A total of 302 women with PPROM were included. Transabdominal amniocentesis was performed and amniotic fluid was assessed. Based of microbial invasion of the amniotic cavity and intra-amniotic inflammation (interleukin-6 ≥ 3000 pg/mL), the women were divided into following groups: intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid. Colonization was found in 11% (32/302) of the women. The most common bacteria identified in the amniotic fluid were  PPROM is characterized by a breach in the barrier between the intra-amniotic and vaginal/cervical environments4. Therefore, PPROM can become complicated by the ascension of microorganisms from the vagina/cervix, thus leading to microorganisms in the amniotic cavity, termed microbial invasion of the amniotic cavity9. Microorganisms in the amniotic fluid are recognized by specialized pattern recognition receptors localized on the amniotic epithelium or in the amniotic fluid13. The activation of these receptors triggers a well-orchestrated intra-amniotic inflammatory response leading to the elevation of the concentrations of various inflammatory mediators (intra-amniotic inflammation), along with the attraction of immune cells into the amniotic fluid, with the ultimate goal of solving the microbial threat16.Preterm prelabor rupture of the membranes (PPROM) is defined as a rupture of the fetal membranes, with leakage of amniotic fluid before the onset of regular uterine activity earlier than 37 weeks of gestation16, with preterm labor with intact membranes17, and those with a short cervix18. This condition is typically considered as either: (1) contamination of the amniotic fluid with skin bacteria during amniotic fluid sampling and/or during pre-analytical processing of amniotic fluid samples or (2) colonization of the amniotic cavity (colonization) with microorganisms from the lower genital tract in the absence or presence of a weak intra-amniotic inflammatory response20.Aside from the "classical" scenario, called intra-amniotic infection, in which the presence of microorganisms in amniotic fluid is associated with intra-amniotic inflammation, an alternative situation, where the presence of microorganisms in the amniotic fluid is not related to intra-amniotic inflammation, has been previously reported among women with PPROM21 and (2) microbial invasion of the amniotic cavity seems to be a consequence of PPROM22.The cause of microbial invasion of the amniotic cavity without intra-amniotic inflammation in PPROM is still under debate; however, colonization is prioritized because (1) the breach in the barrier between the lower genital tract and the amniotic cavity might enhance the ascension of vaginal/cervical bacteria into the amniotic fluid21, colonization is yet to be fully characterized. Therefore, it is essential to fill this knowledge gap and thoroughly investigate the clinical characteristics and significance of colonization to help clinicians deal with PPROM complications.In contrast with intra-amniotic infection and sterile intra-amniotic inflammation in PPROM pregnanciesTherefore, the main aims of this study were (1) to characterize the microbial composition of amniotic fluid from PPROM with colonization, (2) to evaluate the main clinical characteristics of colonization, and (3) to describe the short-term morbidity of newborns from women with colonization.Overall, 302 women with singleton pregnancies with PPROM were included in this study. The clinical definitions are shown in Table p = 0.27). The most common microorganism identified in the amniotic fluid from women with colonization and intra-amniotic infection was Ureaplasma spp. . Women with colonization had a lower load of Ureaplasma spp. DNA in the amniotic fluid than women with intra-amniotic infection  and those with sterile intra-amniotic inflammation .Gestational age at rupture of membranes differed among the subgroups of the women (p = 0.76). However, women with colonization had a shorter latency interval than women with sterile intra-amniotic inflammation . Comparisons with women with intra-amniotic infection were not made due to a different management approach (active management beyond the 28th week of gestation).Latency interval (hours) from rupture of membranes to delivery in women with preterm prelabor rupture of membranes with respect to intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid is shown in Fig. p < 0.0001; Fig. p = 0.04; Fig. Concentrations of interleukin (IL)-6 in amniotic fluid differed among the subgroups of the women . No differences in maternal inflammatory response were found in the comparisons between women with sterile intra-amniotic inflammation  and negative amniotic fluid .Women with colonization had a lower intensity of maternal inflammatory response measured by C-reactive protein (CRP) and white blood cell (WBC) count than women with intra-amniotic infection  of the women (five missing findings were from women with negative amniotic fluid). The rates of acute histological chorioamnionitis (HCA), funisitis and inflammation of the amnion differed among the subgroups of the women  the gestational age at rupture of membranes in women with colonization was similar to that of women with negative amniotic fluid but higher than in those with intra-amniotic inflammation; (4) the intensity of intra-amniotic inflammatory response (IL-6 concentrations) was higher in women with colonization than in those with negative amniotic fluid; (5) the rates of acute inflammatory placental lesions was higher in women with colonization than in those with negative amniotic fluid; and (6) short-term neonatal morbidity did not differ between newborns from PPROM with colonization and those with negative amniotic fluid.The principal findings of this study are as follows: (1) colonization was found in 11% of PPROM pregnancies; (2) 20. The rates of these phenotypes might differ among cohorts of women with PPROM due to differences in distribution of gestational age at rupture of membranes, race/ethnicity, and latency interval between ruptured membranes and sampling. In this study, the rates of intra-amniotic infection and colonization were almost equal . The rate of colonization found in this study was similar to that reported previously (12%) in the study by Romero et al.16; however, the rate of intra-amniotic infection was lower than that reported previously (12% vs. 29%)16. These results show that colonization can be responsible for a significant amount (30–50%) of cases with microbial invasion of the amniotic cavity in PPROM pregnancies. This observation is of clinical significance. However, we must be aware that cases with colonization remain clinically hidden unless the assessment of both microbial invasion of the amniotic cavity and intra-amniotic inflammation is employed. This is in direct contrast to cases with intra-amniotic infection , for which a less expensive and less time-consuming assessment of intra-amniotic inflammation is sufficient.Microbial invasion of the amniotic cavity represents a condition with two clinical phenotypes: (1) intra-amniotic infection when intra-amniotic inflammation is present and (2) colonization/contamination when intra-amniotic inflammation is absentUreaplasma spp. are the most common pathogens in the amniotic fluid from PPROM23. These low virulent commensal bacteria, commonly found in the vagina and/or cervix are among the smallest self-replicating microorganisms that can grow independently26. In this study, the rate of Ureaplasma spp. in women with colonization reached 60%. In addition, a vast majority of microorganisms, other than Ureaplasma spp., identified in the amniotic fluid from this subset of women, were bacteria commonly present in the vagina, cervix, or rectal niche. These observations support that ascension of the bacteria from the lower genital tract might cause colonization in women with PPROM.There is solid evidence that Ureaplasma spp. varies from hundreds to billions of copies of DNA per milliliter of amniotic fluid in PPROM and it is higher in women with intra-amniotic infection than in those with colonization21. Accordingly, a lower burden of Ureaplasma spp. was observed in women with colonization than in those with intra-amniotic infection in this study. Given the dose-dependent relationship between intra-amniotic inflammatory response (measured by IL-6 concentrations) and the amniotic fluid burden of Ureaplasma spp. in PPROM pregnancies28, it is likely that women with colonization, caused by Ureaplasma spp., could not elicit an intra-amniotic inflammatory response intense enough to pass a clinical threshold for intra-amniotic inflammation. In addition, clinicians should be aware of the following facts: (1) intra-amniotic inflammation is expressed as a categorical condition (present/absent) and (2) IL-6 is a physiological constituent of amniotic fluid  compared to those with intra-amniotic infection, sterile intra-amniotic inflammation, and negative amniotic fluid, in which the ranges were almost two-fold higher. These findings might extend our previous observation, where an intensive intra-amniotic inflammatory response to amniotic fluid bacteria, characterized by concentrations of multiple inflammatory-related proteins in amniotic fluid, was not found beyond gestational 32 weeks29.There is evidence that the rate of intra-amniotic infection in PPROM pregnancies decreases with advanced gestational age31. This condition is typically followed by the attraction of neutrophils towards the amniotic cavity from the intervillous space of the placenta into the chorionic plate and/or from the decidua into fetal membranes, leading to the development of acute inflammatory lesions in the placenta32. In this study, both intra-amniotic inflammatory responses, measured by concentrations of IL-6 in the amniotic fluid and the presence of acute inflammatory placental lesions, were assessed. Collectively, women with colonization had a higher intensity of intra-amniotic inflammatory response and rates of acute inflammatory placental lesions than women with negative amniotic fluid. This observation further supports the hypothesis mentioned above that colonization in PPROM represents an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response that is not intense enough to pass a clinical threshold for intra-amniotic inflammation.The intra-amniotic inflammatory response is associated with elevated concentrations of various cytokines, chemokines, and other inflammation-related proteins and lipids in the amniotic fluid33. To extend this knowledge, the selected aspects of short-term neonatal morbidity were investigated in this study. After adjusting for gestational age at delivery, no differences were found between newborns from PPROM with colonization and negative amniotic fluid.It remains debatable whether the microbial invasion of the amniotic cavity and/or intra-amniotic inflammation, after correction for gestational age at delivery, is associated with worse neonatal outcomesUreaplasma spp., M. hominis, and Ch. Trachomatis; non-specific PCR for 16S rRNA; and aerobic/anaerobic cultivation were employed in this study. Finally, short-term neonatal outcomes were available for all newborns in the PPROM cohort.This study had several strengths. First, a relatively large cohort of women with singleton pregnancies complicated by a well-defined clinical phenotype of spontaneous preterm labor (PPROM) with available information about the intra-amniotic environment and histopathology of the placenta was used. Second, a thorough assessment of microbial invasion of the amniotic cavity consisting of specific PCR for Ureaplasma spp. DNA, but not all bacterial DNA, in the amniotic fluid were evaluated in this study. This shortcoming prevented us from assessing whether the burdens of amniotic fluid bacteria other than Ureaplasma spp. differ between women with colonization and those with intra-amniotic inflammation. However, in a recent study on women with preterm labor with intact membranes, microbial and fungal burdens in the amniotic fluid were lower in those with microbial invasion of the amniotic cavity without intra-amniotic inflammation than in those with intra-amniotic inflammation17. Last, the long-term outcomes of infants from PPROM pregnancies were not available.This study also has limitations that are worth mentioning. First, this study consisted of a homogeneous population of Caucasian women living in the eastern part of the Czech Republic. This prevents the findings of this study from being generalized to populations with racial/ethnic disparities. Second, only loads of In conclusion, colonization in PPROM, caused mainly by microorganisms from the lower genital tract, might represent an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response that is not intense enough to pass a clinical threshold for intra-amniotic inflammation.This retrospective study included all pregnant women admitted to the Department of Obstetrics and Gynecology of the University Hospital Hradec Kralove, Czech Republic, between December 2018 and July 2021, who met the following criteria: (1) age ≥ 18 years, (2) PPROM between gestational ages 24 + 0 and 36 + 6 weeks, (3) singleton pregnancy, and (4) transabdominal amniocentesis. The exclusion criteria were as follows: (1) pregnancy-related and other medical complications , (2) congenital or chromosomal fetal abnormalities, (3) signs of fetal hypoxia, and (4) significant vaginal bleeding.Gestational age was determined based on a first-trimester ultrasound scan. PPROM was diagnosed based on visual confirmation of amniotic fluid pooling in the posterior vaginal fornix using a sterile speculum for examination, or by determination of the presence of insulin-like growth factor-binding protein in the vaginal fluid  if uncertainty remained after the clinical examination.Maternal blood and amniotic fluid samples were obtained at the time of admission before the administration of corticosteroids, antibiotics, or tocolytics. The performance of transabdominal amniocentesis to assess the intra-amniotic environment has been a part of the department's standard clinical management of women with PPROM. Women with PPROM were further managed based on amniotic fluid test results. Those with confirmed intra-amniotic inflammation received intravenous clarithromycin for seven days unless delivery occurred. Women without intra-amniotic inflammation were treated with intravenous benzylpenicillin  for seven days unless delivery occurred. Once the final results regarding microbial invasion of the amniotic cavity from cultivation and/or PCR were known, the attending clinician decided to modify the antibiotic therapy. Corticosteroids (betamethasone) were administered to those at gestational ages between 24 + 0 and 34 + 6 weeks to accelerate lung maturation. Tocolysis (atosiban) was used in those who developed regular uterine activity during the course of corticosteroid therapy or within 24 h after their administration.Women with proven intra-amniotic infection beyond the 28th gestational week were managed actively . The remaining women with PPROM were managed expectantly.After delivery, the placenta, fetal membranes, and umbilical cord were fixed in 10% neutral buffered formalin and sent for histopathological evaluation.This study was approved by the approved by the Institutional Review Board of the University Hospital Hradec Kralove . All experiments were performed in accordance with relevant guidelines and regulations. All participants in the study were Caucasian. Informed consent was obtained from all participants.34, cervical fluid samples35, and clinical and demographical data37 from some women from this cohort were used in our previous studies. Therefore, microbial results from amniotic fluid were shown, in part, in these publications37.Amniotic fluid samplesUreaplasma spp., Mycoplasma hominis, and Chlamydia trachomatis; 16S rRNA gene; and aerobic/anaerobic cultivation. The remaining amniotic fluid was further processed, and aliquots and pellets were frozen and used for research purposes. Details about amniotic fluid sampling were described in our previous publication20.During free-hand ultrasonography-guided amniocentesis, performed at the time of admission before administration of corticosteroids, antibiotics, or tocolytics, approximately 3 mL of amniotic fluid was aspirated and used for the assessment of IL-6 concentration; PCR for 38. The measurable range was 1.5–5000 pg/mL, which could be extended to 50,000 pg/mL with a tenfold dilution of the sample. The coefficients of variation for the inter-assay and intra-assay precisions were both < 10%.The concentration of IL-6 in the amniotic fluid was assessed using the automated electrochemiluminescence immunoassay method with the immuno-analyzer Cobas e602 C. trachomatis/Ureaplasma/M. hominis-FRT kit  was used to detect the DNA of Ureaplasma spp., M. hominis, and Ch. trachomatis using a single PCR tube for each fluid. The details have been described previously40. The level of Ureaplasma spp. (copies/mL) was determined using an absolute quantification technique that uses an external calibration curve. Plasmid DNA  was used to prepare the calibration curve.A commercial AmpliSens® 41. The details have been described previously40. The bacteria were then typed using the sequences obtained from BLAST® and SepsiTestTM BLAST20.Bacterial DNA was identified by PCR targeting the 16S rRNA using the following primers: 5-CCAGACTCCTACGGGAGGCAG-3 (V3 region) and 5-ACATTTCACAACACGAGC-GACGA-3 (V6 region)Gardnerella vaginalis selective medium, MacConkey agar, Neisseria-selective medium (modified Thayer–Martin medium), Sabouraud agar, and Schaedler anaerobe agar. The details have been described previously39.The amniotic fluid samples were cultured on Columbia agar with sheep's blood, 43.A maternal blood sample was obtained by venipuncture of the cubital vein and sent to the laboratory immediately following sampling to assess the CRP and WBC count. Details about maternal blood CRP and WBC count are described in our previous publications44. Histopathological examinations were performed by a single pathologist (HH) who was blinded to the clinical status of the women.Tissue samples were obtained from the placenta (at least two samples), fetal membranes , and umbilical cord , which were routinely processed and embedded in paraffin. Sections of the tissue blocks were stained with hematoxylin and eosin. The degree of neutrophil infiltration was evaluated separately in the free membranes (amnion and chorion-decidua), chorionic plate, and umbilical cord according to the criteria provided by Salafia et al.45.Maternal and perinatal medical records were reviewed by five investigators . Data regarding short-term neonatal morbidity were reviewed for all the newborns. Definitions of selected aspects of short-term neonatal morbidity have been described in our previous publicationsUreaplasma spp., M. hominis, C. trachomatis, or their combination; positivity for the 16S rRNA gene; positivity for aerobic/anaerobic cultivation of the amniotic fluid; or a combination of these parameters. Intra-amniotic inflammation was defined as an amniotic fluid with an IL-6 concentration of greater than or equal to 3000 pg/mL46. Colonization was defined as the presence microbial invasion of the amniotic cavity without intra-amniotic inflammation. Intra-amniotic infection was defined as the concurrent presence of microbial invasion of the amniotic cavity and intra-amniotic inflammation. Sterile intra-amniotic inflammation was defined as the presence of intra-amniotic inflammation without microbial invasion of the amniotic cavity. Negative amniotic fluid was defined as amniotic fluid without intra-amniotic inflammation and microbial invasion of the amniotic cavity. The intra-amniotic inflammatory response was characterized by the concentration of IL-6 in the amniotic fluid. Maternal inflammatory response was determined by the concentrations of CRP concentrations and WBC counts in maternal blood. HCA was diagnosed based on the histological grades 3–4 for the chorion-decidua, and/or grades 3–4 for the chorionic plate, and/or grades 1–4 for the umbilical cord, and/or grades 1–4 for the amnion44. Funisitis was diagnosed based on histological grades 1–4 for the umbilical cord44. Inflammation of the amnion was diagnosed based on histological grades 1–4 for the amnion44. Compound neonatal morbidity was defined as the need for intubation, and/or respiratory distress syndrome, and/or transient tachypnea of newborns, and/or bronchopulmonary dysplasia, and/or retinopathy of prematurity, and/or intraventricular hemorrhage, and/or necrotizing enterocolitis, and/or intestinal perforation, and/or early-onset sepsis, and/or late-onset sepsis, and/or neonatal death before hospital discharge20.Microbial invasion of the amniotic cavity was determined based on positive PCR analysis for U tests for continuous variables, as appropriate, and Fisher's exact or chi-square tests for categorical variables, as appropriate, and results were presented as median (interquartile range [IQR]) and number (%), respectively. The normality of the data was tested using the Anderson–Darling test. The loads of Ureaplasma spp. DNA in the amniotic fluid were not normally distributed; therefore, the non-parametric Mann–Whitney U test was used for the analyses. Kaplan–Meier survival curves were constructed, and a log-rank (Mantel-Cox) test was used to compare survival distributions among women with colonization, sterile intra-amniotic inflammation, and negative amniotic fluid. Spearman's partial correlation was used to adjust the results for gestational age at delivery. Differences were considered significant at P < 0.05. All p-values were determined using two-tailed tests, and all statistical analyses were performed using GraphPad Prism v8 for Mac OS X  and the Statistical Package for the Social Sciences (SPSS), version 28.0.0.0, for Windows .The demographic and clinical characteristics of the patients were compared using the non-parametric Kruskal–Wallis or Mann–Whitney Supplementary Information.


The present work shows that the inverse magnetic fluid has advantages such as low cost, no scattering effect, stable fluorescence intensity, and relatively low magnetic resistance. In the end, a prototype design for the novel detection of coronavirus disease 2019 based on the magnetic field induced self-assembly in the inverse magnetic fluid is proposed, which could support the epidemic prevention and control.In the present study, the magnetic field induced self-assembly processes of magnetic microparticles in an aqueous liquid (the pure magnetic fluid) and nonmagnetic microparticles in ferrofluid (the inverse magnetic fluid) are experimentally investigated. The microparticles are formed into chain-like microstructures in both the pure magnetic fluid and the inverse magnetic fluid by applying the external magnetic field. The fluorescence parameters of these self-assembled chain-like microstructures are measured and compared to those without the effect of magnetic field. It is found that the fluorescence in the pure magnetic fluid is weakened, because the scattering and illuminating areas are reduced in the microstructures. On the contrary, the fluorescence in the inverse magnetic fluid is enhanced, because more fluorescent nonmagnetic microparticles are enriched and become detectable under the effect of the magnetic dipole force and the magnetic levitational force, and their unnecessary scattering can be absorbed by the surrounding ferrofluid. The average enhancement of the fluorescence area ratio in the inverse magnetic fluid with 3  I.Since the outbreak of atypical pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was first reported in December 2019, the novel coronavirus disease (COVID-19) was spreading rapidly and showed a great threat to global public health.3O4) microparticles in the phosphate buffer saline (PBS).The IgM and IgG detection tests for human blood or oral mucosal transudate take 15–20 min, and both the ideal sensitivity and the ideal specificity of the detection tests could theoretically reach 100%. However, the real sensitivity and the real specificity of the detection tests on the clinical diagnosis of COVID-19 are only around 80% and 90%, respectively.et al.et al.et al.et al.et al.To approach these problems, a technique of noncontact manipulation in micro/nanoscale, i.e., the magnetic field induced self-assembly in magnetic multiphase flows,The purpose of this work is to investigate the effect of the magnetic field induced self-assembly process on the fluorescence in magnetic multiphase flows and propose a novel detection for COVID-19 based on the magnetic field induced self-assembly technique. To compare the two self-assembly processes in the pure magnetic fluid and the inverse magnetic fluid and reveal their underlying mechanisms, a series of experiments for magnetic and nonmagnetic microparticles are performed. All the self-assembly experiments are observed under an optical microscope with a uniform magnetic field. The remainder of this article is organized as follows. In Sec. II.A.3O4—polystyrene microparticles into deionized water (the common formula in the currently existing antibody detections) and the other is the inverse magnetic fluid by dispersing the nonmagnetic polystyrene microparticles into magnetic fluid (the suspension of magnetic Fe3O4 nanoparticles). The microparticles are synthesized by Bionano New Material Science , and their characterizations are shown in The polystyrene microparticles are commonly used in the biomedicine and medical tests because of their unique advantages, such as good plasticity and durability, well-distributed particle diameter, and uniform shape with a good biocompatibility. Two types of magnetic multiphase fluids are used in this work: one is the pure magnetic fluid by dispersing the core-shell magnetic Feμm nonmagnetic microparticles, 3 μm nonmagnetic fluorescent microparticles, 5 μm magnetic microparticles, and 5 μm magnetic fluorescent microparticles obtained by the Helium Ion Microscopy  are shown in The surface topographies of 3 3O4 nanoparticles are produced by the coprecipitation method which includes two stages. In the first stage, the 40 ml iron (III) chloride solution  and 10 ml magnesium nitrate solution  are mixed, and then added into 350 ml sodium hydroxide solution . After 5 min of heating and stirring, the red–brown precursors [FeOOH/Mg(OH)2] are gradually precipitated. In the second stage, the 400 ml iron (II) chloride solution  is added into the red–brown precursors under an intensive agitation with temperature of 70 °C, and then the mixed solution turns black immediately. The corresponding chemical reaction formulation reads3O4) nanoparticles are obtained. Further, the magnetic Fe3O4 nanoparticles are dispersed into the deionized water with surfactant.In the present study, the magnetic FeB.3 mT = 1 × 104 Gs. H) and the current of DC power supply (A). The snapshots and videos of the magnetic field induced self-assembly in the antibody detection are magnified and observed by the digital metallographic microscope  and recorded by the electronic ocular of the metallographic microscope . The CCD camera used in the present work has 2748 × 2200 pixels.The experimental setup to observe the self-assembly process in the antibody detection are shown in C.3O4 nanoparticles should be prepared with two steps. First, a homogenous ferrofluid is prepared by dissolving the magnetic Fe3O4 nanoparticles in deionized water with 50% alcohol and 0.1% Tween-20 by using an ultrasonic homogenizer. The saturation magnetization of the ferrofluid is around 20 mT (200 Gs), and thus the external magnetic field should be set beyond 20 mT. Then, the nonmagnetic polystyrene microparticles are dispersed in the magnetic fluid by a stirring process or ultrasonic homogenizer. The concentrations of the test magnetic multiphase fluids are shown in Before the experiment, the test magnetic multiphase fluid comprising of the nonmagnetic polystyrene microparticles and the magnetic Feμl test magnetic multiphase fluid from evaporating on the chip. The distance between the slide and coverslip (the thin layer) is around 9.5 μm. When applying the magnetic field, the magnetic field induced self-assembly process could be observed by the CCD camera and recorded by the laptop. Several observation areas of the experimental chip should be recorded and analyzed to obtain the average results. In this work, each test multiphase fluid is measured 2–3 times, and the effective data are obtained from over 10 observation areas. Self-assembly process starts immediately after applying an external magnetic field, and the stable chain-like microstructures are formed after 5 s. Thus, all measurements of self-assembled microstructures begin after 10 s to ensure reliable data. To quantitatively describe the fluorescence in the test magnetic multiphase fluids, the integrated fluorescence density, the fluorescence area, the mean fluorescence intensity, and the fluorescence area ratio are measured in this work.The required strength of the external magnetic field is adjusted by the precision programable DC power supply cooperated with the Tesla meter. The current of DC power supply is set to 3.5 A, and the corresponding strength produced by the electromagnets is 32.1 mT. Then, the experimental chip, i.e., the microscopic glass slide with a coverslip (24 mm × 24 mm), is put into the objective table immediately to prevent the thin layer of the 5.5 III.In this section, the two types of the test magnetic multiphase fluids, i.e., the pure magnetic fluids . The distribution of magnetic microparticles and the fluorescent image under 365 nm ultraviolet light at the initial time are presented in 3O4 core could not be perfectly ensured at the center of the microparticles, and the bulk of adhered microparticles shows a negative effect on the self-assembly process. A typical example of the self-assembly process of decentration microparticles is demonstrated in the blue rectangular region of 3O4 cores in μm magnetic microparticles in the B06 test magnetic multiphase fluid (the pure magnetic fluid) are shown in 3O4 core still exist. Only some weak motions of the magnetic microparticles can be observed under the external magnetic field, which is shown in The magnetic field induced self-assembled microstructures of 5 μm nonmagnetic microparticles in the C06 test magnetic multiphase fluid (the inverse magnetic fluid) is shown in 3O4 nanoparticles. The fluorescence area ratios of The magnetic field induced self-assembly process of 3 μm magnetic microparticles in the D06 test magnetic multiphase fluid (the inverse magnetic fluid). The initial distribution of the nonmagnetic microparticles and the corresponding fluorescent image are shown in μm magnetic microparticles, the boundary layer effects of the slide and coverslip, and the larger distance between two neighboring microparticles caused by the relative lower number density. With these effects, the microparticles move slowly and could be magnetized well at the initial stage of self-assembly. The fluorescence area ratios of B.μm and 5 μm magnetic microparticles. The main reason is that the magnetic microparticles without surface modification of europium are illuminated by the neighboring magnetic fluorescent microparticles due to their scattering effect of the surrounding transparent liquid. The number density of the magnetic microparticles decreases when the magnetic fluorescent microparticle ratio is 80%, and thus fluorescent density is dominated by the magnetic fluorescent microparticles. This effect increases with the microparticle diameter.In this subsection, the integrated fluorescence density, the fluorescence area, the mean fluorescence intensity, and the fluorescence area ratio of the pure magnetic fluid and the inverse magnetic fluid are experimentally quantified. 3O4 nanoparticles.Unlike the pure magnetic fluid, the integrated fluorescence density of the inverse magnetic fluid is monotonically increased due to the magnetic field induced self-assembly. The self-assembled chain-like microstructures gather the nonmagnetic fluorescent microparticles, which can enhance the fluorescence area. Moreover, the integrated fluorescence density of the inverse magnetic fluid is lower than that of the pure magnetic fluid. The main reason is that the fluorescence of the magnetic fluorescence microparticles scatters and illuminates in the surrounding transparent liquid, but the fluorescence of the nonmagnetic fluorescence microparticles is absorbed by the surrounding black Feμm and 5 μm nonmagnetic microparticles are 112.92% and 25.57%, respectively.The average values of fluorescence area and fluorescence area ratio of the test magnetic multiphase fluids are presented in It should be noticed that the mean fluorescence intensity in the present work is not equal to the optical density, and thus the unit of the mean fluorescence intensity is arbitrary. C.3O4 nanoparticles of the inverse magnetic fluid can absorb the redundant light scattered from the nonmagnetic fluorescent microparticles. Third, the magnetic field induced self-assembly process in the inverse magnetic fluid can enhance the fluorescent effect, and the fluorescence intensity of relatively smaller nonmagnetic microparticles is more stable.To quantitatively detect and assay the antibody, it requires a stable fluorescence intensity which should not change with the fluorescent microparticle ratio. The comprehensive analysis of the above experimental results indicates that the inverse magnetic fluid possesses some advantages in the antibody detection. First, the cost of nonmagnetic microparticles is cheaper than that of the magnetic microparticles, and the size of the nonmagnetic microparticles is easily controlled. Second, the magnetic Feϕ is the magnetic potential, r and θ are the radial and angular coordinates, respectively. By applying the method of separation of variable, the analytical solution of the magnetic field intensity inside the circular cylinder can be obtained aspμ) is higher than the magnetic permeability of the base fluid (fμ). The magnetic resistance of the base fluid impedes the conduction of the magnetic field. On the contrary, the magnetic permeability of the base fluid is much higher than that of nonmagnetic microparticle in the inverse magnetic fluid, and the magnetic resistance of the base fluid is very low since the ferrofluids are superparamagnetic.3 smaller than that of the pure magnetic fluid. Therefore, the magnetic field intensity at the center of nonmagnetic microparticle in the ferrofluid is relatively higher than that at the center of magnetic microparticle in the aqueous liquid. The magnetic intensity of the external magnetic field required in the self-assembly process of the inverse magnetic fluid is lower than that of the pure magnetic fluid.Moreover, the magnetic field intensity at the center of nonmagnetic microparticle in the inverse magnetic fluid is higher than that of magnetic microparticles in the pure magnetic fluid. The magnetic potential equation for the circular cylinder under a uniform magnetic field can be written asAccording to these advantages mentioned above, the detection process for COVID-19 based on the magnetic field induced self-assembly in the inverse magnetic fluids is presented in et al.The present fluorescence enhancement in the inverse magnetic fluid by applying the external magnetic field is validated in the quiescent solution. In fact, most existing methods of the antibody detection require a motionless flow field or slow flow field, which is restricted by the frame rate of the camera . To maintain the cost, the fps of the camera is not high. Therefore, to capture the clear pictures of fluorescence, the flow velocity of the test sample during the detection process should be slow. Actually, the self-assembled microstructures can be also formed in Poiseuille flow as demonstrated by Zhang IV.μm nonmagnetic microparticles are increased by employing the magnetic field induced self-assembly, with the average ratio of 112.92%. Moreover, the scattering of fluorescent nonmagnetic microparticle is absorbed by the surrounding ferrofluid, which can provide a quantitative result to retrospect the infection history. This study confirms that the inverse magnetic fluids have following advantages: low cost, no scattering effect, stable fluorescence intensity, and relatively low magnetic resistance. According to these advantages, a prototype design for the novel detection of COVID-19 based on the magnetic field induced self-assembly in the inverse magnetic fluids is proposed, which could support the epidemic prevention and control.Magnetic multiphase flows widely occur in microfluidics, drug delivery, and targeted drugs. The magnetic field induced self-assembly in the magnetic multiphase fluid can be regarded as one of the efficient ways to construct the specific structure in microscale and nanoscale. To investigate the effect of magnetic field induced self-assembly on fluorescence in magnetic multiphase flow, the experimental setup based on the optical microscope is built, and the relative materials are prepared. The surface topographies of the original microparticles and the fluorescent microparticles are observed by using HIM, and the corresponding fluorescence responses are compared under a 365 nm ultraviolet light. Then, a series of experiments by using the magnetic microparticles in aqueous liquid and the nonmagnetic microparticles in ferrofluid are carried out. The self-assembled chain-like microstructures in two kinds of magnetic multiphase flows, i.e., the pure magnetic fluids and the inverse magnetic fluids, are experimentally investigated. The fluorescence parameters of these microstructures are measured and compared with that without the effect of magnetic field, such as the integrated fluorescence density, the fluorescence area, the mean fluorescence intensity, and the fluorescence area ratio. It is found that the fluorescence in the pure magnetic fluids is weakened by the self-assembly process, because the scattering and illuminating areas are reduced in the self-assembled chain-like microstructures. However, the fluorescence is enhanced by the self-assembled chain-like microstructures under the effect of the magnetic dipole force and the magnetic levitational force during the self-assembly process in the inverse magnetic fluid, especially for the nonmagnetic microparticles with smaller diameter. The fluorescence area ratios of 3


We present the ophthalmic findings of a 57-year-old female with pseudoxanthoma elasticum who was initially diagnosed in her 20s after a biopsy of abnormal neck lesions. She began to develop visual problems decades later, but currently has no other related medical problems, including any evidence of coronary artery disease. Our patient complained of bilateral metamorphopsia but with preserved visual acuity of 20/25 in both eyes. In addition to the angioid streaks, the patient also had evidence of pigmentary changes at the posterior pole [Figures 1A–1B]. Optical coherence tomography imaging [Figures 1C–1F] revealed the presence of bilateral subretinal fluid, shaggy photoreceptors, and intraretinal hyperreflective material concentrated within the outer retina that was stable for five years [Figures 1 E & 1F]. Vision is typically not affected by angioid streaks unless patients develop choroidal neovascularization with resultant macular edema and retinal hemorrhages. Following our patient for 10 years , she has never had any evidence of a choroidal neovascular complex on fluorescein angiography and thus it was concluded that the subretinal fluid was the result of malfunctioning retinal pigment epithelium due to the pigmentary changes at the posterior pole, a rare finding first described by Zweifel and colleagues in 2011, who concluded the fluid was due to a similar pathophysiology as pattern dystrophy. Clinicians should be aware of the ophthalmic manifestations of pseudoxanthoma elasticum which include angioid streaks which can lead to choroidal neovascularization and severe vision loss but also pigmentary changes at the posterior pole that can lead to chronic subretinal fluid and a milder effect on visual acuity.Angioid streaks are breaks within a weakened Bruch's membrane. They are deep to the neurosensory retina and typically bilateral, irregular, and emanate from the optic disc. Causes include Ehler–Danlos syndrome, Paget's disease, sickle cell, and other hemoglobinopathies, idiopathic, and pseudoxanthoma elasticum.Nil.There are no conflicts of interest.


Sepsis involves a dysregulated inflammatory response to infection that leads to organ dysfunction. Early fluid resuscitation has been advocated by the Surviving Sepsis Campaign guidelines. However, recent studies have shown that a positive fluid balance is associated with increased mortality in septic patients. We investigated if haemoglobin levels on admission to the intensive care unit (ICU) could modify the association of fluid balance with mortality in patients with sepsis. We hypothesized that with increasing fluid balance, patients with moderate anemia (hemoglobin 7-10g/dL) would have poorer outcomes compared to those without moderate anemia (hemoglobin >10g/dL).This retrospective study utilized the Medical Information Mart for Intensive Care-III (MIMIC-III) database. Patients with sepsis, as identified by the International Classification of Diseases, 9th, Clinical Modification codes, were studied. Patients were stratified into those with and without moderate anemia at ICU admission. We investigated the influence of fluid balance measured within 24 hours of ICU admission on 28-day mortality for both patient groups using multivariable logistic regression models. Subgroup and sensitivity analyses were conducted.8,132 patients  were included. Increasing fluid balance (in L) was associated with a significantly decreased risk of 28-day mortality in patients without moderate anemia . Conversely, increasing fluid balance was associated with a significantly increased risk of 28-day mortality in patients with moderate anemia . Interaction analyses showed that mortality was highest when haemoglobin decreased in patients with moderate anemia who had the most positive fluid balance. Multiple subgroups and sensitivity analyses yielded consistent results.In septic patients admitted to ICU, admission hemoglobin levels modified the association between fluid balance and mortality and are an important consideration for future fluid therapy trials. Sepsis involves a dysregulated inflammatory response to infection that leads to organ dysfunction. It is associated with high mortality and morbidity in patients , 2. In eAlthough fluid administration can help expand intravascular volume and improve perfusion pressure, it is unclear if the iatrogenic hemodilution effect of resuscitation fluids could lead to poorer oxygen carriage and delivery causing fluid administration to paradoxically worsen tissue hypoperfusion. This effect could be made worse in patients with lower hemoglobin levels. A retrospective cohort study of the ARISE trial  found eaet al in adults , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.(2) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: NoReviewer #3: No**********5. Review Comments to the AuthorReviewer #1: The manuscript seeks to elucidate the impact of admission value of hemoglobin on the association between fluid balance and 28-day mortality in sepsis.Major concerns:1. Hemoglobin concentrations are referred to as "admission values", but time points for blood sampling are not specified. Page 11, lines 197-198 exclusion criteria: "174 without hemoglobin measurements in the first 3 days", does that mean that admission hemoglobin value is analyzed sometime during the first 72 h? Please, clarify as it seems important in order to be able to interpret results. Add to/modify accordingly in the Methods, (Results), Discussion.2. p 16, line 261. The choice to present data in tertiles could be discussed and explained. The spread within tertiles is wide.3. p 8, line 140 - 141 "Total fluid balance was winsorized at the 0.5 and 99.5 percentiles to limit the effect of extreme values", Please, give rational for why are data winsorized?4. Figure 3a. tertile 1 has a negative fluid balance with the range -31,25 - 0,61. The authors might wish to discuss and explain how such an extreme negative fluid balance is possible in early sepsis and how these data should be interpreted. In fig 3b range of hemoglobin change in the third tertile (with increasing hemoglobin) is 5 - 285,7 %. This does seem peculiar as the group was defined as non-anemic. Are data correctly reported?5. p9, line 157-159: "Total fluid balance during ICU stay was summarized as a continuous variable in baseline statistics and treated as a dichotomous variable  in further modelling." Please, give rational for dichotomizing the data. The spread/variance of fluid balance over the first 24 hours as depicted in Figure 3a seems extreme. How do the investigators explain negative fluid balance of 32 L?6. The manuscript might benefit from general revision of the language.Minor concerns:p 6, line 76 "It is associated with high mortality and morbidity in survivors" please reformulatep 6, line 82, please comment further on the two references mentioned.p 6, line 94 "duration of ventilation, length of stay" please specifyp 7, line 110-111 "have poorer outcomes in the first 24 hours", please correctReviewer #2: Thank you for allowing me to review “Association of fluid balance with mortality….” By Sandra Tan et al. Here, the purpose was to examine whether low hemoglobin (Hb) levels are associated with a poorer outcome of sepsis treatment. There are undoubtedly many patients studied, 9,700, and the included data are from admission to the ICU only (not before), the fluid balance during the first 24 h, and the mortality 28 days later. The results hold that positive fluid balance was associated with decreased 28-day mortality if Hb levels were on the high side. The reverse was the case in patients with low Hb levels (< 100 g/L).My problem is that I don´t know if we learn anything from the results. Hb levels can be modified by many factors in severe disease. Inflammation leads to capillary leakage that raises Hb. If septic shock has occurred spontaneous capillary refill can be expected to having reduced the Hb concentration. Patients in bad condition and a low arterial pressure are likely to receive more IV fluid than others, which further reduces Hb.We don't know in what clinical situation the patients were in when the “admission Hb” was taken. Had the patients already received i.v. fluid a bolus 30 mL/kg? Had they low MAP, which causes hemodilution by capillary refill? Had they waited a long time before entering the ICU? The analysis gets more complicated as Hb varies greatly among populations, and even within populations. Normal range in this country is 110-165 g/L.If the authors can clearly point out the clinical implication of their results I am willing to change my judgment. Could it be that infusing much fluid on the first day of care is beneficial is Hb is high? If so, it would at least be logical on a simple level in a complex diseaseReviewer #3: Tan et al have investigated the association between admission hemoglobin levels and positive fluid balance influenced mortality in ICU patients.1. The article lacks certain novelty, and the results need to be revised deeply; firstly, the amount of fluid balance is not clear in the definition of variables. Early fluid resuscitation in different ICU groups must be adjusted according to patients’ hemodynamic parameters. The more positive fluid balance doses have a greater impact on the prognosis of patients. I will suggest that the author can make a stratified analysis between different fluid balance and different hemoglobin levels to clarify the influence of hemoglobin levels on the prognosis of ICU patients during fluid therapy.2. The definition of moderate anemia with hemoglobin 7-10g/dl is not properly, for hemoglobin lower than 8g/dl may influence patients’ outcome. The author needs to stratify different levels of hemoglobin, not just roughly with 7-10g/dl.3. ICU patients’outcome can influence by many different parameters. It is difficult to exclude the mixing factors using logistical regression, and the results lack credibility. In this paper, to confirm the connection between positive fluid balance and hemoglobin levels in mortality, all the confound factors need to be strictly matched. At the same time, the statistical analysis needs to be further improved.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  15 May 2021Please see attached file titled 'Response to Reviewers'.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 19 May 2021Association of fluid balance with mortality in sepsis is modified by admission hemoglobin levels: a large database studyPONE-D-21-00459R1Dear Dr. Tan,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Aleksandar R. ZivkovicAcademic EditorPLOS ONE 27 May 2021PONE-D-21-00459R1 Association of fluid balance with mortality in sepsis is modified by admission hemoglobin levels: a large database study Dear Dr. Tan:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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To assess glioblastoma multiforme (GBM) formation with similar imaging characteristics to human GBM using multiparametric magnetic resonance imaging (MRI) in an orthotopic xenograft canine GBM model.The canine GBM cell line J3T1 was subcutaneously injected into 6-week-old female BALB/c nude mice to obtain tumour fragments. Tumour fragments were implanted into adult male mongrel dog brains through surgery. Multiparametric MRI was performed with conventional MRI, diffusion-weighted imaging, and dynamic susceptibility contrast-enhanced perfusion-weighted imaging at one week and two weeks after surgery in a total of 15 surgical success cases. The presence of tumour cells, the necrotic area fraction, and the microvessel density (MVD) of the tumour on the histologic specimen were assessed. Tumour volume, diffusion, and perfusion parameters were compared at each time point using Wilcoxon signed-rank tests, and the differences between tumour and normal parenchyma were compared using unpaired t-tests. Spearman correlation analysis was performed between the imaging and histologic parameters.P > 0.05). There was greater cerebral blood volume and flow in the GBM than in the normal-appearing white matter . The MVD in the histologic specimens was correlated with the cerebral blood volume in the GBM tissue .All animals showed a peripheral enhancing lesion on MRI and confirmed the presence of a tumour through histologic analysis (92.3%). The normalized perfusion values did not show significant decreases through at least 2 weeks after the surgery (Our results suggest that the canine GBM model showed perfusion imaging characteristics similar to those of humans, and it might have potential as a model to assess novel technical developments for GBM treatment. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumour and carries a poor prognosis . The pooHowever, it is challenging to apply the knowledge obtained from the rodent models widely used in preclinical studies to human GBM. The small animal GBM models’ limitation is that they are too small to evaluate newly applied diagnostic or treatment devices –6. The cin vivo physiological status, such as the abnormal blood vessel formation with higher cerebral blood volume (CBV) observed in human GBM [The orthotopic xenograft canine model may yield physiologic information about the tumour itself through the multiparametric MRI used in current clinical imaging due to having sufficient brain size for image acquisition , 7. The uman GBM –15.Therefore, this study aimed to assess GBM formation with similar imaging characteristics to human GBM using multiparametric MRI and pathological results in an orthotopic xenograft canine GBM model.This study was approved by the Institutional Animal Care and Use Committee of the Seoul National University Hospital  and was performed in accordance with the Guide from our IACUC and the National Institute of Health Guide for the Care and Use of Laboratory Animals. All animals were maintained in an AAALAC International (#001169) accredited facility in accordance with Guide for the Care and Use of Laboratory Animals 8th edition, NRC (2010).The canine glioblastoma cell line J3T1, derived from the canine glioma cell line J3T, was provided by the Okayama University School of Medicine, School of Dentistry, after obtaining research ethics approval. The cell lines were maintained in Roswell Park Memorial Institute (RPMI) medium with 10% fetal bovine serum (FBS) and a 1% penicillin/streptomycin mixture  at 37°C. Cell viability was assessed with trypan blue staining to confirm a cell viability of > 90% before tumour implantation.5/100 μL medium/mouse) were subcutaneously injected into the flanks of 6-week-old female BALB/c nude mice . After 2 weeks, the mice were sacrificed with a lethal dose of sodium pentobarbital  to obtain tumour fragments. The tumour fragments were excised to a diameter of 3–5 mm, washed with phosphate-buffered saline (PBS), and suspended in 50% basement membrane matrix  in an icebox.The experimental design is summarized in Eighteen healthy male mongrel dogs  were used in this study and were purchased from the International Laboratory Animal Center . They were housed separately in stainless steel cages (W 895 × L 795 × H 765 mm) in an environmentally controlled room . Food and sterilized water were available ad libitum.Three immunosuppressive agents were used to suppress the immune system in the experimental dogs before tumour implantation. A regimen based on one previously used canine gliosarcoma and cavernous sinus tumour model was modified and optimized through preliminary experiments , 17. TheThe surgical procedure for canine tumour implantation was performed under general anaesthesia. The animals were sedated with intravenous administration of a 1:1 combination of tiletamine hydrochloride and zolazepam  and xylazine hydrochloride  at a concentration of 5 mg/kg. Then, respiratory anaesthesia was maintained with a ventilator using 0.5 to 3% isoflurane during the surgical procedure.All procedures were performed under aseptic techniques after disinfecting the right frontoparietal region and covering it with a sterilized cloth. A craniotomy was performed to create a small burr hole (0.3–0.5 cm in diameter) using a handpiece drill . CSF pulsation in the subdural space was confirmed through the hole, and the prepared tumour fragment was implanted using an 18-gauge spinal needle. Procedural bleeding was prevented and controlled using bone wax  and N-butyl cyanoacrylate . The overlaying muscle and skin were then closed using a 3–0 Vicryl suture and a 4–0 nylon suture. All dogs were given intramuscular injections of cephazolin  to prevent infection for 7 days, and the surgical sites were disinfected daily for 7 days after surgery.MRI scans were performed using a 3.0 T MR imaging system  with a human head coil. All MRI examinations were performed with the dogs in the supine position and included the entire brain. The animals were sedated with the same intravenous sedative drug administration method as used for the surgery during all MRI examinations.The MRI sequences included coronal turbo-spin echo T2-weighted imaging (T2WI), coronal gradient-echo T1-weighted imaging (T1WI), and coronal contrast-enhanced T1WI (CE-T1WI). CE T1WI was performed after an intravenous injection of 0.2 mL/kg gadoteric acid  via the cephalic vein. MRI parameters were as follows: 5160/91 ms/131°/640 × 290/5 mm (TR/TE/FA/matrix/section thickness) for T2WI and 990/9.8 ms/70°/384 × 222/1.5 mm for T1WI and CE-T1WI.2, 38 sections, a 3-mm section thickness, a 0.9-mm intersection gap, an FOV of 280 × 280 mm, a matrix of 192 × 192, and three signal averages [Diffusion-weighted imaging (DWI) was obtained with a single-shot spin-echo echo-planar imaging sequence in the coronal plane before contrast injection, similar to the imaging method in humans , 18. DWIaverages . An averaverages .DSC-PWI was acquired in the same way as that used in humans using a single-shot gradient-echo imaging sequence during the contrast agent’s intravenous injection , 18, 19.Image acquisition was performed at baseline before tumour implantation and every week after surgery until sacrifice. The dogs were assigned to one of the two groups with one-week and two-week follow-ups because of gingival hypertrophy due to long-term use of cyclosporine for more than two weeks in our preliminary experiment.The image analysis methods are presented in One investigator  drew the regions of interest (ROIs) that contained the entire tumour on every continuous section of the coregistered images. Tumour boundaries were defined with reference to the high-signal intensity areas thought to represent tumour tissue on CE-T1WI. The ROIs included all contrast-enhancing areas, and the values were automatically obtained with the software by summing up all values from each coregistered axial slice, which were then averaged. After obtaining the total voxel values of the rCBV of each tumour, the mean rCBV of each tumour was calculated. In addition, the rCBV values in the normal-appearing white matter contralateral to the enhancing lesion and in the cerebellum were measured for comparison with the rCBV of the tumour. On a pixel-by-pixel basis, the normalized CBV (nCBV) maps were calculated by dividing every relative CBV value by those for unaffected white matter using the NordicICE software package, and all of the pixels from the entire relative CBV map were normalized relative to the mean value of the ROI placed on the contralateral normal-appearing white matter, resulting in the nCBV map. For the comparison of perfusion status between tumours and normal-appearing white matter, the nCBV values, which were calculated on the tumour and normal-appearing white matter by dividing the cerebellum’s value, were obtained and then compared . Other pAll of the dogs were sacrificed with an overdose of intravenous pentobarbital sodium . The specimens were fixed in 10% neutral buffered formalin for one week and then sliced along the coronal plane for a full examination. The specimens were embedded in paraffin and cut into 4-μm sections. Each section was stained with Harris’ haematoxylin solution and eosin Y (H&E)  to assess the presence of tumour cells and changes in the vasculature. A pathologist  who was blinded to the experimental information reviewed the specimens. The presence of tumour cells was assessed first, and then the necrotic area fraction (%) from the H&E-stained sections was calculated by dividing the area of the largest cross-section by the area of necrosis in a lower-power field (×40). To obtain the microvessel density (MVD) of the tumour, hot spots, i.e., areas of higher vascular density than the rest of the tissue, were chosen in three random fields per individual tumour section in a low-power field (×40), from which the vessels were counted at high magnification (×400).P-value < 0.05 was considered statistically significant. The Wilcoxon signed-rank test was utilized to evaluate the differences in tumour volume, normalized ADC values, and normalized perfusion values on the 2-week follow-up MRI. Additionally, unpaired Student’s t-tests were performed to compare the ADC and perfusion values between the tumour and normal-appearing white matter. Spearman correlation analysis was performed between the perfusion parameters and MVD.All statistical analyses were performed using a commercial software program . A The results of the development of the orthotopic xenograft canine GBM model are summarized in P = 0.043). Neither the nCBV nor nTTP value showed significant serial decreases through the 2-week follow-up imaging . However, there was a slight decrease in the nCBF and an increase in the nMTT value at the 2-week follow-up (P = 0.043 and P = 0.043). Additionally, there was no significant difference in the nADC value among the follow-up images (P = 0.500).The serial follow-up imaging characteristics are summarized in P < 0.001 and P < 0.001, respectively). However, a significant difference was not observed between the GBM and normal groups in either the ADC or nADC values.The differences in perfusion and diffusion parameters in the GBM and normal-appearing white matter are summarized in A representative case is shown in P = 0.004). However, the percentage of the necrotic portion did not correlate with the normalized perfusion parameters or the ADC value .To further confirm the presence of GBM, we performed histopathological analysis . The numIn the present study, we evaluated the imaging characteristics of the canine brain tumour model through multiparametric MRI. The human GBM-like imaging characteristics were validated through the increase in the CBV and CBF accompanied by a histopathological reference using the MVD in the canine GBM model. Therefore, it might be a possible preclinical model to assess novel diagnostic and treatment methods for human GBM.Rodent GBM models made by intracranial or subcutaneous implantation are widely used in GBM studies and are advantageous for their high tumorigenesis and reproducible growth rate . HoweverSeveral attempts have been made to establish a reproducible model of human GBM in large animals. Whelan et al.  reportedThe MRI features of human GBM typically show a large necrotic mass with thick and irregular enhancing margins with peritumoural oedema on conventional sequences . HoweverIn our study, there was higher CBV and CBF in the canine GBM than in the normal-appearing white matter, which is similar to the imaging findings in human GBM. On serial follow-up MRI, the tumour’s peripheral enhancement, although reduced in size, remained for at least two weeks. It may be reasonable to assume the presence of a tumour given the persistent irregular enhancement and increase in CBV and CBF, which was also related to histopathological findings, despite the decreasing size of the enhancement. DSC-PWI is known to estimate the tissue MVD by measuring the CBV . Our canHowever, in terms of the ADC values, we could not demonstrate a significant difference between the GBM and normal-appearing white matter tissue or in the nADC values on the serial follow-up images. The ADC values may reflect tumour cellularity, with higher values in cystic areas than in the solid component of the tumour . The lacSeveral limitations of the present study should be mentioned. First, we could not assess the appropriate blood concentration of cyclosporine in the development and maintenance of the induced tumours in the canine GBM model. Additionally, long-term data from more than two weeks postsurgery could not be obtained because of cyclosporine-induced gingival hypertrophy. Precise dose adjustment during the follow-up period is needed. However, an extended follow-up period might not be necessary, especially in the case of experiments to evaluate the potential of novel materials and device technologies for human GBM . The canIn conclusion, this study showed that the canine GBM model exhibited perfusion imaging characteristics similar to those of humans and histopathological features to human GBM. The canine GBM model can help clinicians understand the pathophysiology of GBM, and we believe that it can be used as a potential experimental model for diagnostic and therapeutic assessment for GBM.


Currently, approximately 150 different brain tumour types are defined by the WHO. Recent endeavours to exploit machine learning and deep learning methods for supporting more precise diagnostics based on the histological tumour appearance have been hampered by the relative paucity of accessible digital histopathological datasets. While freely available datasets are relatively common in many medical specialties such as radiology and genomic medicine, there is still an unmet need regarding histopathological data. Thus, we digitized a significant portion of a large dedicated brain tumour bank based at the Division of Neuropathology and Neurochemistry of the Medical University of Vienna, covering brain tumour cases from 1995–2019. A total of 3,115 slides of 126 brain tumour types (including 47 control tissue slides) have been scanned. Additionally, complementary clinical annotations have been collected for each case. In the present manuscript, we thoroughly discuss this unique dataset and make it publicly available for potential use cases in machine learning and digital image analysis, teaching and as a reference for external validation. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.16652272 Overall, they are relatively uncommon neoplasms with an incidence of approximately 24 per 100.000 person-years3. Current diagnostic guidelines published by the WHO define approximately 150 distinct brain tumour types and assign grades I to IV, based on malignancy and potential to malignant transformation or progression. They are mainly differentiated by their histopathological phenotypes and molecular alterations4. While the majority of tumours is diagnosed solely based on histopathology, an integrated approach is mandatory for 19 tumour types.Brain tumours account for a large fraction of years of potential life lost as compared with tumours from other sites5. Recently, diagnostic algorithms trained on DNA methylation data have been shown to significantly increase diagnostic accuracy6. Similar advances focusing on histopathological data have been hampered, so far, by the lack of freely available histopathology datasets7. Most available histopathology data such as those available through TCGA8, IvyGAP10 or TCIA11 focus on only a few diagnostic entities. They mostly consist of digitized fresh frozen tissue sections, which feature relatively poor tissue morphology as compared to formalin-fixed and paraffin-embedded tissues. Still, even with these limited data, computational algorithms have been successfully trained - amongst others - for survival prediction12, detection of tumour-infiltrating lymphocytes13, and assessments of tumour microvessels14. However, larger datasets encompassing an even wider range of brain tumours and featuring improved cellular and morphological characteristics are necessary to further develop these algorithms and extend their applicability to the entire spectrum of brain tumour types.Still, more accurate diagnostic distinctions are needed in order to i) better assess individual patients’ prognoses and ii) support more robust therapeutic decisionsThus, we set out to compile a comprehensive resource of digitized Haematoxylin-eosin(H&E)-stained brain tumour whole slide images (WSIs) with clinical annotations Fig. . We aime15.H&E stained tumour slides from FFPE tissues, which were collected for routine diagnostics in the time interval of 1995–2019 have been obtained from the biobank of the Division of Neuropathology & Neurochemistry, Medical University of Vienna. We digitized each slide in high magnification  using a Hamamatsu NanoZoomer 2.0 HT slide-scanner. Each slide was manually reviewed to ensure high scan quality and sufficient diagnostic tumour tissue. Samples with equivocal diagnoses or missing molecular work-up otherwise needed to assign an integrated WHO 2016 diagnosis were excluded. A subset of glioblastoma scans (n = 381) has been published previously as part of the GBMatch studyBasic clinical annotations consisting of patient age and sex as well as tumour location and recurrence were acquired from local electronic records where available. Tumour locations have been assigned to the following 19 categories: frontal; parietal; insular; occipital; temporal; cerebellar; brain stem; spinal; lateral ventricle; diencephalon; third ventricle; fourth ventricle; sellar region; cranial nerves; basal ganglia; cerebral, NOS (not otherwise specified); posterior fossa, NOS; cranial, NOS; and other.This study complies with the relevant ethical, legal and institutional regulations and the study protocol has been approved by the Ethics Committee of the Medical University of Vienna (EK1691–2017). Participant informed consent has been obtained as by institutional guidelines, necessitating restrictions on commercial use of the obtained data.16. In summary, H&E stained WSIs were first colour-deconvoluted into separate Haematoxylin and Eosin channels17. Then, global, Phansalkar and Otsu thresholding were applied to the Haematoxylin channel to identify nuclei19. Watershedding was used to separate densely clustered cells20. Only cells with a minimum size of 4 pixels were kept. The total tissue area was determined by averaging all colour channels, thresholding at a threshold of 220, followed by binary close and open operations.Additionally, the total tissue area and the average cellularities were estimated for each scan using a custom MATLAB script  with a similar approach as previously published21 as one ndpi-file per sample, sorted by diagnostic tumour type  for easier access. It is possible to download single files directly or all files of a specific tumour type or the whole dataset using a download manager (such as the Chrono Download Manager for the Google Chrome browser). Furthermore, supplementary clinical information, estimated cell densities and scanned tissue area is provided in a csv-spreadsheet with one row per tumour sample. An overview of all spreadsheet variables and descriptions is given in Table Data are provided via EBRAINSA total of 3,115 histological slides of 2,880 patients have been scanned. A total of 126 distinct diagnostic tumour types could be included. There are 1,395 female and 1,462 male patients in the dataset. The mean patient age at brain tumour surgery was 45 years, ranging from 9 days to 92 years. 2,530 of the scanned slides originated from primary operations and 538 from re-operations. See online-only Table All cases were initially selected based on the given diagnosis in the diagnostic electronic records. To ensure conformity with the WHO 2016 diagnosis, all slides have been independently reviewed by two neuropathologists experienced in neuro-oncology. In disputed cases, a third senior neuropathologist was consulted. Older cases with missing necessary molecular analyses were not included in the dataset.Inter- and intraobserver variability is one factor that contributes to misdiagnoses or discrepant diagnoses. We mitigated the risk by including only cases that had already undergone thorough routine diagnostic work-up and were additionally reviewed independently by at least two neuropathologists as described above. In this way, we also ensured excellent image quality and the presence of sufficient diagnostic tumour tissue on each WSI. Scans with suboptimal image quality were either re-scanned (if possible) or excluded.21. In order to download the data set, users have to register with EBRAINS and agree to the general terms of use, access policy as well as the data use agreement for pseudonymised human data (https://ebrains.eu/terms). The data are distributed under the conditions that users cite the respective DOI, adhere to EBRAINS’ Data Use Agreement and do not use the data for commercial purposes.The data can be accessed via EBRAINS22. Alternatively, most other WSI programs such as the open-source OMERO software platform23 and the open-source QuPath software24 can work directly on ndpi-files. However, most programming languages and non-specialized image processing software cannot handle ndpi-files out of the box. Thus, we also provide a toolbox of MATLAB scripts that depend on the openslide library25 and can be used toAutomatically tile large slide scans and export multiple smaller image patches in a given magnification.Convert annotation-files (.ndpa) to overlays, which can be used to extract specific regions of interest.Estimate the total tissue area on a WSI.Estimate the cell density on a WSI.The ndp.view2 (© Hamamatsu) software can be freely used to view and annotate slide scans saved in the ndpi format26, more recent methods employing generative adversarial networks27 or style transfer learning28. Moreover, heavy stain colour augmentation should be performed29. Of note, the stain normalization step can be omitted with only a negligible drop in performance as has been shown by Tellez et al.29.Of note, slide thickness and staining intensity vary to some degree, resulting in a slightly different histological appearance of each slide. Thus, for machine learning applications, we recommend astain normalization step such as WSICS