Automated Flow Cytometer Research Articles

Point-of-care neutrophil CD64 as a rule in diagnostic test for bacterial infections in the emergency department

IntroductionBacterial infections are frequently seen in the emergency department (ED), but can be difficult to distinguish from viral infections and some non-infectious diseases. Common biomarkers such as c-reactive protein (CRP) and white blood cell (WBC) counts fail to aid in the differential diagnosis. Neutrophil CD64 (nCD64), an IgG receptor, is suggested to be more specific for bacterial infections. This study investigated if nCD64 can distinguish bacterial infections from other infectious and non-infectious diseases in the ED.MethodsAll COVID-19 suspected patients who visited the ED and for which a definitive diagnosis was made, were included. Blood was analyzed using an automated flow cytometer within 2 h after presentation. Patients were divided into a bacterial, viral, and non-infectious disease group. We determined the diagnostic value of nCD64 and compared this to those of CRP and WBC counts.ResultsOf the 291 patients presented at the ED, 182 patients were included with a definitive diagnosis (bacterial infection n = 78; viral infection n = 64; non-infectious disease n = 40). ROC-curves were plotted, with AUCs of 0.71 [95%CI: 0.64–0.79], 0.77 [0.69–0.84] and 0.64 [0.55–0.73] for nCD64, WBC counts and CRP, respectively. In the bacterial group, nCD64 MFI was significantly higher compared to the other groups (p < 0.01). A cut-off of 9.4 AU MFI for nCD64 corresponded with a positive predictive value of 1.00 (sensitivity of 0.27, a specificity of 1.00, and an NPV of 0.64). Furthermore, a diagnostic algorithm was constructed which can serve as an example of what a future biomarker prediction model could look like.ConclusionFor patients in the ED presenting with a suspected infection, nCD64 measured with automatic flow cytometry, has a high specificity and positive predictive value for diagnosing a bacterial infection. However, a low nCD64 cannot rule out a bacterial infection. For future purposes, nCD64 should be combined with additional tests to form an algorithm that adequately diagnoses infectious diseases.

Intermittent Upwelling Events Trigger Delayed, Major, and Reproducible Pico‐Nanophytoplankton Responses in Coastal Oligotrophic Waters

AbstractPico‐nanophytoplankton organisms are dominant in oceanic oligotrophic areas but their adaptive growth rates make their contribution to the carbon cycle difficult to estimate. Here we address their response capacities after sporadic wind gusts causing upwelling events in a coastal Mediterranean station. When the water column is stratified, corresponding to oligotrophic conditions, these events generate intense short‐lived nutrient pulses and seawater temperature drops lasting 6 days on average with decreases up to 10°C. Using an automated flow cytometer and statistical rupture‐detection methods, we characterize the responses of five pico‐nanophytoplankton functional groups at a two‐hour frequency from September 2019 to November 2021. These events trigger delayed increases in both abundances and biomasses following similar patterns for most groups that can overpass spring bloom values, and are immediately followed by an overall decrease, suggesting a clear physical driver. These submesoscale events, due to their short duration, are poorly represented in coastal carbon budgets.

Leukocyte cell population data in patients with cardiac surgery and cardiopulmonary bypass: A potential readily available tool to monitor immunity.

Cardiac surgery with cardiopulmonary bypass triggers sterile inflammation that is responsible for post-operative morbidity. Automated flow cytometry devices used for leucocyte count provide cell population data (CPD) regarding fluorescence intensity, size and granularity of leukocytes that have never been studied in the context of sterile inflammation. Our objective was to explore leukocyte cell population data in patients undergoing cardiac surgery with cardiopulmonary bypass in order to determine whether CPD could be used to monitor immune cell activation. This is an ancillary study of a cohort of patients undergoing cardiac surgery with cardiopulmonary bypass. Cell population data (CPD) extracted from a routine automated flow cytometer were analyzed (Fluorescence targeted to nucleic acids). The time points of interest were: pre-operative, postoperative and 5 days after surgery. The variations in those parameters were studied. Data were then compared between patients according to the occurrence of a composite criteria (supra-ventricular arrythmia, stroke, acute renal failure, and/or death). Data from 1453 patients were analyzed. The neutrophil count, fluorescence granularity (NE-SCC), intensity (NE-SFL) and size (NE-FSC) increased with surgery. Heterogeneity of neutrophils decreased in terms of fluorescence granularity (NE-WX) and size (NE-WZ) but increased in terms of intensity (NE-WY). The lymphocyte count decreased with surgery. While fluorescence granularity (LY-X) and size increased (LY-Z), Lymphocyte intensity decreased (LY-Y). Lymphocytes were less heterogeneous in terms of their granularity, size and intensity after surgery (LY-WX, LY-WY, LY-WZ). Patients who developed the composite complication criteria had a higher pre-operative neutrophil count (5.08 [3.89;6.95] vs 4.76 [3.60;6.13], p = 0.02; AUC = 0.56 [0.51;0.60]), and more heterogeneous neutrophils in terms of fluorescence granularity (NE-WX, AUC = 0.57 [0.52;0.62]) and intensity (NE-WY, AUC 0.61 [0.56;0.65]). Those patients also had lower pre-operative lymphocyte count (1.49 [1.10;1.14] vs 1.81 [1.39;2.39], p<0.01, AUC = 0.61 [0.57;0.66]) and fluorescence granularity (LY-X, AUC = 0.57 [0.53;0.62]). NE-WX, NE-WY and LY-X were associated with post-operative complications after adjustment on the EuroSCORE 2 (adjusted odd ratio of 1.01 [1.00;1.02]; 1.01 [1.00;1.01] and 1.08 [1.02;1.15] respectively). Cardiac surgery with cardiopulmonary bypass was associated with substantial alterations of CPD probably reflecting leukocytes activation in sterile inflammation. Pre-operative NE-WX, NE-WY and LY-X biomarkers levels were associated with post-operative complications, independently of the EuroSCORE 2. Such routine, unexploited and low cost parameters might represent useful tools likely to monitor immune function and predict outcomes for patients undergoing cardiac surgery. Our findings requires validation on a larger external cohort.

Karenia selliformis bloom dynamics and growth rate estimation in the Sfax harbour (Tunisia), by using automated flow cytometry equipped with image in flow, during autumn 2019

A Karenia selliformis bloom event in the Gulf of Gabès (Mediterranean Sea), was monitored over 9 days at high frequency during fall 2019, by using an automated flow cytometer (Cytosense, Cytobuoy b.v.) with an image-in-flow attachment. The instrument recorded the shape of the optical signals that lead to the resolution of six cell groups of pico-, nano- and microphytoplankton, during the Harmful Algal Bloom (HAB). K. selliformis cell dimensions derived from the hourly records, enabled to estimate the daily division rate over the bloom period. Results revealed that K. selliformis was the only bloom-forming species and it reached its highest mean abundance the fourth day of the survey. A shift in the nutrient composition occurred with a potential P limitation during the bloom growth and N limitation during the bloom collapse. The co-inertia analysis revealed opposite patterns for K. selliformis and heterotrophic prokaryotes suggesting trophic interactions and possible mixotrophic behaviour of K. selliformis at the end of the bloom. K. selliformis exhibited low growth rates generally < 1 division day−1, which could not explain the observed high abundance. The tide played a crucial role in the dynamics of K. selliformis at a semi-diurnal scale and at spring-neap tide scale and was probably enhancing K. selliformis accumulation.

NEUT-SFL in Patients with COVID-ARDS: A Novel Biomarker for Thrombotic Events?

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped RNA virus first identified in December 2019 in Wuhan, China, and responsible for coronavirus disease 2019 (COVID-19). The ongoing COVID-19 pandemic is impacting healthcare worldwide. Patients who develop coagulopathy have worse outcomes. The pathophysiology of COVID-19 suggests a strong interplay between hemostasis and immune cells, especially neutrophils. Our purpose was to assess neutrophil fluorescence as a potential biomarker of deep vein thrombosis (DVT) in patients with COVID-acute respiratory distress syndrome (COVID-ARDS). Sixty-one patients with COVID-ARDS admitted to the four intensive care units (ICUs) of a French general hospital were included in this prospective study. Neutrophil activation was assessed by measuring neutrophil fluorescence (NEUT-Side Fluorescence Light, NEUT-SFL) with a specific fluorescent dye staining analyzed by a routine automated flow cytometer Sysmex XN-3000™ (Sysmex, Kobe, Japan). DVT was diagnosed by complete duplex ultrasound (CDU). We found that NEUT-SFL was elevated on admission in patients with COVID-ARDS (49.76 AU, reference value 46.40 AU, p < 0.001), but did not differ between patients with DVT (49.99 AU) and those without (49.52 AU, p = 0.555). NEUT-SFL is elevated in patients with COVID-ARDS, reflecting neutrophil activation, but cannot be used as a marker of thrombosis. Because neutrophils are at interface between immune response and hemostasis through release of neutrophil extracellular traps, monitoring their activation could be an interesting approach to improve our management of coagulopathy during COVID-ARDS. Further research is needed to better understand the pathophysiology of COVID-19 and identify high-performance biomarkers.

Measurement of red blood cell size based on a lensless imaging system.

The variability in the size of red blood cells (RBCs) is an important additional diagnostic parameter for diseases, which has been established as a part of the complete blood count (CBC). The CBC can be performed using an automated flow cytometer, but it is too bulky and expensive for point-of-care testing. A miniaturized lensless imaging system is a competitive modality for a CBC, that is small and inexpensive. There are two challenges in developing a lensless imaging system for taking the CBC, which make the measurement of the RBC size very difficult: the diffraction effect and the low resolution. In this paper, the RBC radius measurement is replaced with a diffraction ring radius measurement. The diffraction ring radius is much larger than the RBC radius. This feature can improve the imaging resolution. Based on Fresnel diffraction, the relationship between the radius of RBCs and the diffraction fringes is analyzed. Finally, a complete measurement algorithm for determining the RBC size based on the lensless imaging system is given, which can be used to measure the variability in the size of RBCs. In our experiment, the maximum error is less than 6.74%.

Point-of-Care Analysis of Neutrophil Phenotypes: A First Step Toward Immuno-Based Precision Medicine in the Trauma ICU.

The amount of tissue damage and the amplitude of the immune response after trauma are related to the development of infectious complications later on. Changes in the neutrophil compartment can be used as read out of the amplitude of the immune response after trauma. The study aim was to test whether 24/7 point-of-care analysis of neutrophil marker expression by automated flow cytometry can be achieved after trauma. A prospective cohort study was performed. Polytrauma patients who developed infectious complications were compared with polytrauma patients who did not develop infectious complications. The study was performed in a level 1 trauma center. All trauma patients presented in the trauma bay were included. An extra blood tube was drawn from all patients. Thereafter, a member of the trauma team placed the blood tube in the fully automated flow cytometer, which was located in the corner of the trauma room. Next, a modified and tailored protocol for this study was automatically performed. The trauma team was able to successfully start the point-of-care automated flow cytometry analysis in 156 of 164 patients, resulting in a 95% success rate. Polytrauma patients who developed infectious complications had a significantly higher %CD16dim/CD62Lbright neutrophils compared with polytrauma patients who did not develop infectious complications (p = 0.002). Area under the curve value for %CD16dim/CD62Lbright neutrophils is 0.90 (0.83-0.97). This study showed the feasibility of the implementation of a fully automated point-of-care flow cytometry system for the characterization of the cellular innate immune response in trauma patients. This study supports the concept that the assessment of CD16dim/CD62Lbright neutrophils can be used for early detection of patients at risk for infectious complications. Furthermore, this can be used as first step toward immuno-based precision medicine of polytrauma patients at the ICU.

P331Effect of exercise stress test on platelet function in patients with recent acute myocardial infarction

Abstract Background Exercise-based cardiac rehabilitation for coronary artery disease (CAD) is associated with lower cardiovascular mortality. On the other hand, acute strenuous exercise has been linked to cardiovascular complications such as acute myocardial infarction (AMI) and sudden cardiac death. One of the pathophysiological mechanisms involved in these outcomes might be an increase in platelet aggregability after exercise. Although previous studies showed higher platelet aggregability after exercise among stable CAD patients on aspirin treatment, there is no data regarding the effect of exercise on platelet activity in post-AMI patients on dual anti-platelet therapy (DAPT). Purpose To evaluate the effect of high-intensity exercise on platelet aggregability in sedentary post-AMI patients on DAPT. Methods Platelet function was analyzed immediately before and after maximal cardiopulmonary exercise test (CPET) on cycle ergometer utilizing a personalized ramp protocol and aiming to achieving peak exercise in around 10 min. The CPET was done within 31±4 days after uncomplicated AMI. Platelet aggregability was assessed by Multiplate®ADPtest (MP-ADP) and Multiplate® ASPItest (MP-ASPI) measured as area under the curve (AUC). Reticulated platelets were measured concomitantly to MP-ADP e MP-ASPI using a fully automated flow cytometer (Sysmex XN-2000®) to determine absolute immature platelet count (IPC) per 103/microliter. Continuous variables were expressed as means ±standard deviation or as median and 25th–75th percentiles if not Gaussian distributed. Comparisons between the pre- and post-CPET assessments were performed using Wilcoxon signed rank test. Results We analyzed 81 sedentary patients (mean age 58.3±10.1 years-old, 76.5% men) after AMI (50.6% with ST-elevation myocardial infarction, mean left ventricular ejection fraction after index event 55±11.7%, 98.8% on statin and 85.5% on beta-blocker treatment). Platelet aggregability, either by MP-ADP or MP-ASPI, and IPC were significantly increased after CPET (table). Platelet function after CPET Before CPET After CPET p-value Multiplate® ADPtest (AUC) – median (25th–75th percentiles) 32.0 (22.0–48.5) 37.0 (26.0–55.2) 0.003 Multiplate® ASPItest (AUC) – median (25th–75th percentiles) 17.0 (12.7–22.0) 22.0 (16.7–28.0) &lt;0.001 Immature platelet count (103/microliter) – median (25th–75th percentiles) 9.5 (6.8–13.8) 9.6 (6.6–16.5) 0.006 CPET: cardiopulmonary exercise test; AUC: area under the curve. Conclusion On this post-AMI population, platelet was hyperactivated after exercise stress test despite the use of DAPT. These findings suggest that, even when properly treated, post-AMI patients might be at higher risk of ischemic complications after high-intensity exercises, reinforcing the importance of tailoring exercise prescription in this population. Acknowledgement/Funding Sao Paulo Research Foundation, FAPESP

Wash-Free Multiplexed Mix-and-Read Suspension Array Fluorescence Immunoassay for Anthropogenic Markers in Wastewater.

Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers. Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer. For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.

Trait‐based analysis of subpolar North Atlantic phytoplankton and plastidic ciliate communities using automated flow cytometer

AbstractPlankton are an extremely diverse and polyphyletic group, exhibiting a large range in morphological and physiological traits. Here, we apply automated optical techniques, provided by the pulse‐shape recording automated flow cytometer—CytoSense—to investigate trait variability of phytoplankton and plastidic ciliates in Arctic and Atlantic waters of the subpolar North Atlantic. We used the bio‐optical descriptors derived from the CytoSense (light scattering [forward and sideward] and fluorescence [red, yellow/green and orange from chlorophylla, degraded pigments, and phycobiliproteins, respectively]) and translated them into functional traits to demonstrate ecological trait variability along an environmental gradient. Cell size was the master trait varying in this study, with large photosynthetic microplankton (&gt; 20 μm in cell diameter), including diatoms as single cells and chains, as well as plastidic ciliates found in Arctic waters, while small‐sized phytoplankton groups, such as the picoeukaryotes (&lt; 4μm) and the cyanobacteriaSynechococcuswere dominant in Atlantic waters. Morphological traits, such as chain/colony formation and structural complexity (i.e., cellular processes, setae, and internal vacuoles), appear to favor buoyancy in highly illuminated and stratified Arctic waters. In Atlantic waters, small cell size and spherical cell shape, in addition to photo‐physiological traits, such as high internal pigmentation, offer chromatic adaptation for survival in the low nutrient and dynamic mixing waters of the Atlantic Ocean. The use of automated techniques that quantify ecological traits holds exciting new opportunities to unravel linkages between the structure and function of plankton communities and marine ecosystems.

The standardization of the report for urine cell counting-A converting factor for Sysmex UF-1000i.

BackgroundMulticenter laboratory may apply both automated flow cytometer and microscopy for urinalysis. Automated flow cytometer such as Sysmex UF‐1000i evaluates particles with native urine without centrifugation and reports as “counts per μL.” Microscopic examination recommended as the reference method for urine sediment analysis reports results as “counts per HPF (or μL).” Moreover, some results from flow cytometer are needed to be checked visually under microscopy. Therefore, it is worth to establish the consistency of the results from these two methods.MethodsUrine specimens from 412 patients were examined with Sysmex UF‐1000i and manual microscopy using FAST‐READ disposable counting chambers. White blood cell (WBC) and red blood cell (RBC) counting results from UF‐1000i after transferred with the converting factor (0.297) we estimated were compared with that from microscopic examination. Method comparison was performed using Passing‐Bablok analysis.ResultsAfter transferred with the converting factor (0.297), cell counting results from UF‐1000i showed a good correlation with that derived by the reference method (R 2 was 0.868 for RBCs (P < 0.001), 0.882 for WBCs (P < 0.001)). Passing‐Bablok analysis showed no systematic difference (intercept estimate, −1 [95%CI, −7 to 3] and slightly proportional (slope estimate, 1.2 [95%CI, 1.0 to 1.7]) bias between concentrations of cells measured by manual microscopy and Sysmex UF‐1000i using the converting factor.ConclusionThe converting factor (0.297) helps to transfer “counts per μL (non‐centrifugal urine)” to “counts per μL (equal to centrifugal urine),” and to keep the urine particle analysis results of Sysmex UF‐1000i consistent with the results from the reference method.

Coupling physics and biogeochemistry thanks to high-resolution observations of the phytoplankton community structure in the northwestern Mediterranean Sea

Abstract. Fine-scale physical structures and ocean dynamics strongly influence and regulate biogeochemical and ecological processes. These processes are particularly challenging to describe and understand because of their ephemeral nature. The OSCAHR (Observing Submesoscale Coupling At High Resolution) campaign was conducted in fall 2015 in which a fine-scale structure (1–10 km∕1–10 days) in the northwestern Mediterranean Ligurian subbasin was pre-identified using both satellite and numerical modeling data. Along the ship track, various variables were measured at the surface (temperature, salinity, chlorophyll a and nutrient concentrations) with ADCP current velocity. We also deployed a new model of the CytoSense automated flow cytometer (AFCM) optimized for small and dim cells, for near real-time characterization of the surface phytoplankton community structure of surface waters with a spatial resolution of a few kilometers and an hourly temporal resolution. For the first time with this optimized version of the AFCM, we were able to fully resolve Prochlorococcus picocyanobacteria in addition to the easily distinguishable Synechococcus. The vertical physical dynamics and biogeochemical properties of the studied area were investigated by continuous high-resolution CTD profiles thanks to a moving vessel profiler (MVP) during the vessel underway associated with a high-resolution pumping system deployed during fixed stations allowing sampling of the water column at a fine resolution (below 1 m). The observed fine-scale feature presented a cyclonic structure with a relatively cold core surrounded by warmer waters. Surface waters were totally depleted in nitrate and phosphate. In addition to the doming of the isopycnals by the cyclonic circulation, an intense wind event induced Ekman pumping. The upwelled subsurface cold nutrient-rich water fertilized surface waters and was marked by an increase in Chl a concentration. Prochlorococcus and pico- and nano-eukaryotes were more abundant in cold core waters, while Synechococcus dominated in warm boundary waters. Nanoeukaryotes were the main contributors (&gt;50 %) in terms of pigment content (red fluorescence) and biomass. Biological observations based on the mean cell's red fluorescence recorded by AFCM combined with physical properties of surface waters suggest a distinct origin for two warm boundary waters. Finally, the application of a matrix growth population model based on high-frequency AFCM measurements in warm boundary surface waters provides estimates of in situ growth rate and apparent net primary production for Prochlorococcus (μ=0.21 d−1, NPP =0.11 mgCm-3d-1) and Synechococcus (μ=0.72 d−1, NPP =2.68 mgCm-3d-1), which corroborate their opposite surface distribution pattern. The innovative adaptive strategy applied during OSCAHR with a combination of several multidisciplinary and complementary approaches involving high-resolution in situ observations and sampling, remote-sensing and model simulations provided a deeper understanding of the marine biogeochemical dynamics through the first trophic levels.

Neutrophil Fluorescence: A New Indicator of Cell Activation During Septic Shock-Induced Disseminated Intravascular Coagulation.

To investigate the contribution of neutrophil activation as innate immune cells during septic shock-induced disseminated intravascular coagulation. Prospective study. One University Hospital ICU. Hundred patients with septic shock. Thirty-five patients had disseminated intravascular coagulation according to Japanese Association for Acute Medicine 2006 score. None. Neutrophil chromatin decondensation was assessed by measuring neutrophil fluorescence (NEUT-side-fluorescence light) labeled by a fluorochrome-based polymethine reagent using a routine automated flow cytometer Sysmex XN20 (Sysmex, Kobe, Japan) and neutrophil-derived CD66b microparticles by prothrombinase assay. Measurements in disseminated intravascular coagulation and no disseminated intravascular coagulation patients showed that a mean value of NEUT-side-fluorescence light above 57.3 arbitrary units had a sensitivity of 90.91% and a specificity of 80.60% for disseminated intravascular coagulation diagnosis. NEUT-side-fluorescence light was correlated to the CD66b microparticles/neutrophil count, a surrogate of neutrophil activation associated with septic shock-induced disseminated intravascular coagulation. NEUT-side-fluorescence light, routinely available, could prove an accurate biomarker of neutrophil activation.

The Shaggy Ink Cap Medicinal Mushroom, Coprinus comatus (Higher Basidiomycetes) Extract Induces Apoptosis in Ovarian Cancer Cells via Extrinsic and Intrinsic Apoptotic Pathways.

In a previous study, ethyl acetate extract of Coprinus comatus was found to reduce viability of human ovarian cancer cells. The objective of the current research was to clarify the mechanism of action of this extract. Ovarian cancer cells (ES-2) were subjected to ethyl acetate extract of C. comatus for different concentrations or exposure times. Cell cycle analysis and annexin V staining were performed using an automated flow cytometer. DNA fragmentation was detected using the TUNEL assay. Western blot analysis was performed for the assessment of activation of caspases -3, -8, and -9. Results revealed that treatment of ES-2 cells with ethyl acetate extract of C. comatus (100 μg/ml medium), for 48 h or for 72 h resulted in an increased number of cells at the sub-G1 phase of the cell cycle. These treatments also resulted in an increased number of apoptotic cells (positively stained by annexin and positively labeled by TUNEL), in comparison to the control. Reduced levels of procaspases -3, -8, and-9 were also detected in treated cells. In conclusion, ethyl acetate extract of C. comatus induces apoptosis in ovarian cancer cells (ES-2), via both extrinsic and intrinsic pathways. Meanwhile, more investigations are needed to demonstrate weather the apoptotic effect on ovarian cancer cells is accomplished by one active compound, or combined activities of different compounds that exist in the extract.

Clinical role of Notch signaling pathway in intraductal papillary mucinous neoplasm of the pancreas.

This study was performed to elucidate the expression of the Notch signaling pathway and its correlations to clinicopathological factors of intraductal papillary mucinous neoplasms (IPMNs). It is incontrovertible that regulatory T cells (Tregs) play an important role in tumor immunity. However, the whole mechanism of control of peripheral Tregs remains unclear. It is also known that the Notch signaling pathway is involved in Treg suppressor function. Moreover, IPMNs have a high malignant potential. Peripheral blood samples and resected specimens from 18 patients with IPMN were evaluated. All patients were pathologically diagnosed with IPMN. Resected specimens were immunohistochemically evaluated (anti-Notch1, anti-Notch2, and anti-Notch2-intracellular domain antibody staining) and compared in terms of clinicopathological factors. Peripheral Treg populations were analyzed with an automated flow cytometer. Disease-free survival was significantly worse in the Notch1 high-expression group (P = 0.023). Notch2 family expressions were higher in intraductal papillary mucinous carcinoma (IPMC) than in intraductal papillary mucinous adenoma (IPMA) (Notch2, P = 0.012; Notch2-intracellular domain, P = 0.036). Jagged1 expression was significantly higher in IPMC than in IPMA (P < 0.05) and was significantly related to recurrence. The Treg population in peripheral blood was higher in patients with IPMC than in those with IPMA (P < 0.01). Notch signaling, especially Jagged1 expression, reflects IPMN aggressiveness. Our data may suggest that the Notch signaling pathway is a key pathway that determines IPMN pathological aggressiveness and reflects the peripheral Treg population.

Consequence of a sudden wind event on the dynamics of a coastal phytoplankton community: an insight into specific population growth rates using a single cell high frequency approach.

Phytoplankton is a key component in marine ecosystems. It is responsible for most of the marine primary production, particularly in eutrophic lagoons, where it frequently blooms. Because they are very sensitive to their environment, the dynamics of these microbial communities has to be observed over different time scales, however, assessment of short term variability is often out of reach of traditional monitoring methods. To overcome these limitations, we set up a Cytosense automated flow cytometer (Cytobuoy b.v.), designed for high frequency monitoring of phytoplankton composition, abundance, cell size, and pigment content, in one of the largest Mediterranean lagoons, the Berre lagoon (South-Eastern France). During October 2011, it recorded the cell optical properties of 12 groups of pico-, nano-, and microphytoplankton. Daily variations in the cluster optical properties were consistent with individual changes observed using microscopic imaging, during the cell cycle. We therefore used an adaptation of the size-structured matrix population model, developed by Sosik et al. (2003) to process the single cell analysis of the clusters and estimate the division rates of 2 dinoflagellate populations before, during, and after a strong wind event. The increase in the estimated in situ daily cluster growth rates suggest that physiological changes in the cells can prevail over the response of abundance.

Onset of the spring bloom in the northwestern Mediterranean Sea: influence of environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure.

Most of phytoplankton influence is barely understood at the sub meso scale and daily scale because of the lack of means to simultaneously assess phytoplankton functionality, dynamics and community structure. For a few years now, it has been possible to address this objective with an automated in situ high frequency sampling strategy. In order to study the influence of environmental short-term events (nutrients, wind speed, precipitation, solar radiation, temperature, and salinity) on the onset of the phytoplankton bloom in the oligotrophic Bay of Villefranche-sur-Mer (NW Mediterranean Sea), a fully remotely controlled automated flow cytometer (CytoSense) was deployed on a solar-powered platform (EOL buoy, CNRS-Mobilis). The CytoSense carried out single-cell analyses on particles (1–800 μm in width, up to several mm in length), recording optical pulse shapes when analyzing several cm3. Samples were taken every 2 h in the surface waters during 2 months. Up to 6 phytoplankton clusters were resolved based on their optical properties (PicoFLO, Picoeukaryotes, Nanophytoplankton, Microphytoplankton, HighSWS, HighFLO). Three main abundance pulses involving the 6 phytoplankton groups monitored indicated that the spring bloom not only depends on light and water column stability, but also on short-term events such as wind events and precipitation followed by nutrient pulses. Wind and precipitation were also determinant in the collapse of the clusters' abundances. These events occurred within a couple of days, and phytoplankton abundance reacted within days. The third abundance pulse could be considered as the spring bloom commonly observed in the area. The high frequency data-set made it possible to study the phytoplankton cell cycle based on daily cycles of forward scatter and abundance. The combination of daily cell cycle, abundance trends and environmental pulses will open the way to the study of phytoplankton short-term reactivity to environmental conditions.

A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

BackgroundMicrobial cell population heterogeneity is now recognized as a major source of issues in the development and optimization of bioprocesses. Even if single cell technologies are available for the study of microbial population heterogeneity, only a few of these methods are available in order to study the dynamics of segregation directly in bioreactors. In this context, specific interfaces have been developed in order to connect a flow cytometer directly to a bioreactor for automated analyses. In this work, we propose a simplified version of such an interface and demonstrate its usefulness for multiplexed experiments.ResultsA low-cost automated flow cytometer has been used in order to monitor the synthesis of a destabilized Green Fluorescent Protein (GFP) under the regulation of the fis promoter and propidium iodide (PI) uptake. The results obtained showed that the dynamics of GFP synthesis are complex and can be attributed to a complex set of biological parameters, i.e. on the one hand the release of protein into the extracellular medium and its uptake modifying the activity of the fis promoter, and on the other hand the stability of the GFP molecule itself, which can be attributed to the protease content and energy status of the cells. In this respect, multiplexed experiments have shown a correlation between heat shock and ATP content and the stability of the reporter molecule.ConclusionThis work demonstrates that a simplified version of on-line FC can be used at the process level or in a multiplexed version to investigate the dynamics of complex physiological mechanisms. In this respect, the determination of new on-line parameters derived from automated FC is of primary importance in order to fully integrate the power of FC in dedicated feedback control loops.

Pediatric reference values for urine particle quantification by using automated flow cytometer: Results of a multicenter study of Italian urinalysis group

ObjectivesThe purpose of this Italian multicenter study was to define pediatric upper reference values for urine particle quantification by using automated flow cytometry. Design and methodsFour hospital-based clinical laboratories participated in this multicenter investigation, which included a total study population of 161 Italian children aged from 1 to 12years. Two laboratories used Sysmex UF-100 and analyzed 86 children, whereas the other two used Sysmex UF-1000i and analyzed 75 subjects. Particle quantification included the analysis of white blood cells (WBC), red blood cells (RBC), squamous epithelial cells (EC), transitional epithelial cells (TC), casts (CAST) and bacteria (BACT). ResultsThe upper reference values in subjects tested with the Sysmex UF-100 were 9.7WBC/μL, 10.1RBC/μL, 7.5EC/μL, 2.5TC/μL, 0.7CAST/μL and 3090BACT/μL, whereas the upper reference values in subjects tested with the Sysmex UF-1000i were 10.5WBC/μL, 8.3RBC/μL, 7.2EC/μL, 2.9TC/μL, 0.7CAST/μL and 48BACT/μL. No statistically significant differences between genders were found in the value distribution of any of the parameters tested. Similarly, no statistically significant differences were observed between the two urine analyzers, except for BACT. ConclusionsAutomated analysis of urine particles appears a suitable means to optimize the workflow of routine urinalysis of children specimens. The upper reference limits for pediatric subjects obtained in this study were comparable to those previously reported in the literature, with no significant differences between genders and analyzers.

Usefulness of peripheral regulatory T-cells induced by Notch signaling pathway-driven indoleamine 2, 3-dioxygenase positive dendritic cell as a biomarker for the decision for IPMN surgical indications.

181 Background: This study was performed to elucidate the expression of the Notch signaling pathway regulated by dendetric cell (DC) and their correlations to clinicopathological factors of intraductal papillary mucinous neoplasms (IPMNs) as a new biomarker for surgical indications. We already reported that regulatory T cells (Tregs) play an important role in tumor immunity (Pancreas 2006, ASCO-GI 2009), however, the whole mechanism of control of peripheral Tregs remains unclear. It is reported that Indoleamine 2, 3-dioxygenase (IDO) induces active Treg from naïve CD4+Tcells through dendritic cells. Otherwise, we also reported that the Notch signaling pathway is involved in tumor growth and DC function (JI 2010). Thus we focused that Indoleamine 2,3-deoxygenase(IDO)-Treg axis driven by Notch signaling in IPMNs. Methods: Peripheral blood samples and resected specimens from 20 patients with IPMN were evaluated. All patients were pathologically diagnosed with IPMN. Resected specimens were immunohistochemically evaluated (anti-Notch1, anti-Notch2, anti-Notch2-intracellular domain and anti-IDO antibody staining) and compared to clinicopathological factors. Peripheral Treg populations were analyzed with an automated flow cytometer. Results: Disease-free survival was significantly worse in the Notch1 high-expression group (P&lt;0.05). Notch2 family expressions were higher in intraductal papillary mucinous carcinoma (IPMC) than in intraductal papillary mucinous adenoma (IPMA) (Notch2, P&lt; 0.05; Notch2-intracellular domain, P &lt; 0.05). Jagged1 and IDO expressions were significantly higher in IPMC than in IPMA (P &lt; 0.05) and was significantly related to recurrence. The Treg population in peripheral blood was higher in patients with IPMC than in those with IPMA (P &lt; 0.01). Conclusions: Notch signaling, especially Jagged1 expression, regulated by IDO+DC reflects IPMN aggressiveness. Our data strongly suggest that peripheral Treg induced by Notch signaling pathway driven IDO+DC may be a nobel biomarker for the decision for IPMN surgical indications.